This invention relates to novel bicyclic pyrimidine and pyridine derivatives that are useful in the treatment of hyperproliferative diseases, such as cancers, in mammals. This invention also relates to a method of using such compounds in the treatment of hyperproliferative diseases in mammals, especially humans, and to pharmaceutical compositions containing such compounds.
Compounds that are useful in the treatment of hyperproliferative diseases are also disclosed in the following co-pending patent applications: PCT international patent application number PCT/IB97100675 (filed Jun. 11, 1997), U.S. provisional patent application No. 601041846 (filed Apr. 9, 1997), U.S. provisional patent application No. 60/031862 (filed Nov. 27, 1996), U.S. provisional patent application No. 60/028881 (filed Oct. 17, 1996), PCT international patent application number PCT/IB97/00584 (filed May 22, 1997), U.S. patent application Ser. No. 08/653,786 (filed May 28, 1996), PCT international patent application publication number WO 96/40142 (published Dec. 19, 1996), PCT international patent application publication number WO 97/13771 (published Apr. 17, 1997), and PCT international patent application publication number WO 95/23141 (published Aug. 31, 1995). Each of the foregoing United States and PCT international patent applications is incorporated herein by reference in its entirety.
It is known that a cell may become cancerous by virtue of the transformation of a portion of its DNA into an oncogene (i.e. a gene that upon activation leads to the formation of malignant tumor cells). Many oncogenes encode proteins which are aberrant tyrosine kinases capable of causing cell transformation. Alternatively, the overexpression of a normal proto-oncogenic tyrosine kinase may also result in proliferative disorders, sometimes resulting in a malignant phenotype.
Receptor tyrosine kinases are large enzymes that span the cell membrane and possess an extracellular binding domain for growth factors such as epidermal growth factor, a transmembrane domain, and an intracellular portion that functions as a kinase to phosphorylate specific tyrosine residue in proteins and hence to influence cell proliferation. The foregoing tyrosine kinases may be classified as growth factor receptor (e.g. EGFR, PDGFR, FGFR and erbB2) or non-receptor (e.g. c-src and bcr-abl) kinases. It is known that such kinases are often aberrantly expressed in common human cancers such as breast cancer, gastrointestinal cancer such as colon, rectal or stomach cancer, leukemia, and ovarian, bronchial or pancreatic cancer. Aberrant erbB2 activity has been implicated in breast, ovarian, non-small cell lung, pancreatic, gastric and colon cancers. It has also been shown that epidermal growth factor receptor (EGFR) is mutated or overexpressed in many human cancers such as brain, lung, squamous cell, bladder, gastric, breast, head and neck, oesophageal, gynecological and thyroid cancers. Thus, it is believed that inhibitors of receptor tyrosine kinases, such as the compounds of the present invention, are useful as selective inhibitors of the growth of mammalian cancer cells.
It has also been shown that EGFR inhibitors may be useful in the treatment of pancreatitis and kidney disease (such as proliferative glomerulonephritis and diabetes-induced renal disease), and may reduce successful blastocyte implantation and therefore may be useful as a contraceptive. See PCT international application publication number WO 95/19970 (published Jul. 27, 1995).
It is known that polypeptide growth factors such as vascular endothelial growth factor (VEGF) having a high affinity to the human kinase insert-domain-containing receptor (KDR) or the murine fetal liver kinase 1 (FLK-1) receptor have been associated with the proliferation of endothelial cells and more particularly vasculogenesis and angiogenesis. See PCT international application publication number WO 95/21613 (published Aug. 17, 1995). Agents, such as the compounds of the present invention, that are capable of binding to or modulating the KDR/FLK-1 receptor may be used to treat disorders related to vasculogenesis or angiogenesis such as diabetes, diabetic retinopathy, hemangioma, glioma, melanoma, Kaposi""s sarcoma and ovarian, breast, lung, pancreatic, prostate, colon and epidermoid cancer.
The present invention relates to compounds of the formulas 1 and 2 
and to pharmaceutically acceptable salts and hydrates thereof, wherein:
wherein X1 is N or CH;
R1 is H, C1-C6 alkyl or xe2x80x94C(O)(C1-C6 alkyl);
R2 is C6-C10 aryl or 5-13 membered heterocyclic, wherein said R2 groups are optionally substituted by 1 to 5 R5 substituents,
each R5 is independently selected from halo, cyano, nitro, trifluoromethoxy, trifluoromethyl, azido, xe2x80x94C(O)R8, xe2x80x94C(O)OR8, xe2x80x94OC(O)R8, xe2x80x94OC(O)OR8, xe2x80x94NR6C(O)R7, xe2x80x94C(O)NR6R7, xe2x80x94NR6R7, xe2x80x94OR9, xe2x80x94SO2NR6R7, C1-C6 alkyl, xe2x80x94(CH2)jO(CH2)qNR6R7, xe2x80x94(CH2)tO(CH2)qOR9, xe2x80x94(CH2)tOR9, xe2x80x94S(O)j(C1-C6 alkyl), xe2x80x94(CH2)t(C6-C10 aryl), xe2x80x94(CH2)t(5-10 membered heterocyclic); xe2x80x94C(O)(CH2)t(C6-C10 aryl), xe2x80x94(CH2)tO(CH2)j(C6-C10 aryl), xe2x80x94(CH2)tO(CH2)q(5-10 membered heterocyclic), xe2x80x94C(O)(CH2)t(5-10 membered heterocyclic), xe2x80x94(CH2)jNR7(CH2)qNR6R7, xe2x80x94(CH2)jNR7CH2C(O)NR6R7, xe2x80x94(CH2)jNR7(CH2)qNR9C(O)R8, xe2x80x94(CH2)jNR7(CH2)tO(CH2)qOR9, xe2x80x94(CH2)jNR7(CH2)qS(O)j(C1-C6 alkyl), xe2x80x94(CH2)jNR7(CH2)tR6, xe2x80x94SO2(CH2)t(C6-C10 aryl), and xe2x80x94SO2(CH2)t(5-10 membered heterocyclic), wherein j is an integer ranging from 0 to 2, t is an integer ranging from 0 to 6, q is an integer ranging from 2 to 6, the xe2x80x94(CH2)qxe2x80x94 and xe2x80x94(CH2)txe2x80x94 moieties of the foregoing R5 groups optionally include a carbon-carbon double or triple bond where t is an integer between 2 and 6, and the alkyl, aryl and heterocyclic moieties of the foregoing R5 groups are optionally substituted by 1 to 3 substituents independently selected from halo, cyano, nitro, trifluoromethyl, azido, xe2x80x94C(O)R8, xe2x80x94C(O)OR8, xe2x80x94OC(O)R8, xe2x80x94OC(O)OR8, xe2x80x94NR6C(O)R7, xe2x80x94C(O)NR6R7, xe2x80x94(CH2)tNR6R7, C1-C6 alkyl, xe2x80x94(CH2)t(C6-C10 aryl), xe2x80x94(CH2)t(5-10 membered heterocyclic), xe2x80x94(CH2)tO(CH2)qOR9, and xe2x80x94(CH2)tOR9, wherein t is an integer ranging from 0 to 6 and q is an integer ranging from 2 to 6;
each R6 and R7 is independently selected from H, C1-C6 alkyl, xe2x80x94(CH2)t(C6-C10 aryl), xe2x80x94(CH2)t(5-10 membered heterocyclic), xe2x80x94(CH2)tO(CH2)qOR9, and xe2x80x94(CH2)tOR9, wherein t is an integer ranging from 0 to 6 and q is an integer ranging from 2 to 6, and the alkyl, aryl and heterocyclic moieties of the foregoing R6 and R7 groups are optionally substituted by 1 to 3 substituents independently selected from halo, cyano, nitro, trifluoromethyl, azido, xe2x80x94C(O)R8, xe2x80x94C(O)OR8, xe2x80x94CO(O)R8, xe2x80x94OC(O)OR8, xe2x80x94NR9C(O)R10, xe2x80x94C(O)NR9R10, xe2x80x94NR9R10, C1-C6 alkyl, xe2x80x94(CH2)t(C6-C10 aryl), xe2x80x94(CH2)t(5-10 membered heterocyclic), xe2x80x94(CH2)tO(CH2)qOR9, and xe2x80x94(CH2)tOR9, wherein t is an integer ranging from 0 to 6 and q is an integer ranging from 2 to 6, with the proviso that where R6 and R7 are both attached to the same nitrogen, then R6 and R7 are not both bonded to the nitrogen directly through an oxygen;
each R8 is independently selected from H, C1-C10 alkyl, xe2x80x94(CH2)t(C6-C10 aryl), and xe2x80x94(CH2)t(5-10 membered heterocyclic), wherein t is an integer ranging from 0 to 6;
each R9 and R10 is independently selected from H and C1-C6 alkyl; and,
R11 is H, C1-C6 alkyl, xe2x80x94C(O)NR6R9, xe2x80x94C(O)(C6-C10 aryl), xe2x80x94(CH2)t(C6-C10 aryl), or xe2x80x94(CH2)t(5-10 membered heterocyclic), wherein t is an integer ranging from 0 to 6, wherein said R11 groups, other than H, are optionally substituted by 1 to 5 R5 groups.
Preferred compounds include those of formula 1 wherein R11 is xe2x80x94(CH2)t(C6-C10 aryl) or xe2x80x94(CH2)t(5-10 membered heterocyclic), wherein t is an integer ranging from 0 to 6, wherein said R11 groups are optionally substituted by 1 to 5 R5 groups. Specific preferred R11 groups include phenyl or pyridyl, wherein said phenyl and pyridyl are optionally substituted by 1 to 5 R5 groups.
Other preferred compounds include those of formula 1 wherein X1 is CH.
Other preferred compounds include those of formula 1 wherein R2 is phenyl optionally substituted by 1 to 5 R5 substituents, or R2 is a group of the formula 
wherein X2 is xe2x80x94Sxe2x80x94 or xe2x80x94N(R6)xe2x80x94, X3 is N or CH, the dashed line in formula 3 represents an optional double bond, and the above R2 groups of formulas 3 and 5 are optionally substituted by 1 to 5 R5 substituents and the R2 groups of formulas 4 and 6 are optionally substituted by 1 to 3 R5 substituents. Specifically preferred compounds include those wherein R2 is a group of formula 3 above wherein said group is optionally substituted by 1 to 5 R5 substituents.
Specific embodiments of the present invention include the following compounds:
(3-Ethynyl-phenyl)-(6-phenyl-thieno[3,2-d]pyrimidin-4-yl)-amine;
(3-Ethynyl-phenyl)-[6-(4-methoxy-phenyl)-thieno[3,2-d]pyrimidin-4-yl]-amine;
Benzo[b]thiophen-5-yl-(6-phenyl-thieno[3,2-d]pyrimidin-4-yl)-amine;
(1H-Indol-5-yl)-[6-(4-methoxy-phenyl)-thieno[3,2-d]pyrimidin-4-yl]-amine;
(1H-Indol-5-yl)-(6-phenyl-thieno[3,2-d]pyrimidin-4-yl)-amine;
(6-Phenyl-thieno[3,2-d]pyrimidin-4-yl)-(2-pyrrol-1-yl-phenyl)-amine;
(5-Phenyl-1H-pyrazol-3-yl)-(6-phenyl-thieno[3,2-d]pyrimidin-4-yl)-amine;
(5-Phenyl-1H-pyrazol-3-yl)-thieno[3,2-d]pyrimidin-4-yl-amine;
(1H-Indol-5-yl)-thieno[3,2-d]pyrimidin-4-yl-amine;
N-(5-Phenyl)-1-(6-phenyl-thieno[3,2-d]pyrimidin-4-yl)-1H-[1,2,4]triazole-3,5-diamine;
3-[3-Phenyl-5-(6-phenyl-thieno[3,2-d]pyrimidin-4-ylamino)-pyrazol-1-yl]-propionitrile;
(5-Furan-2-yl-2H-pyrazol-3-yl)-(6-phenyl-thieno[3,2-d]pyrimidin-4-yl)-amine;,
(6-Phenyl-thieno[3,2-d]pyrimidin-4-yl)-(5-thiophen-2-yl-2H-pyrazol-3-yl)-amine;
N-(6-Phenyl-thieno[3,2-d]pyrimidin-4-yl)-benzene-1,4-diamine;
N-[4-(6-Phenyl-thieno[3,2-d]pyrimidin-4-ylamino)-phenyl]-benzamide;
N-Methyl-Nxe2x80x2-(6-phenyl-thieno[3,2-d]pyrimidin-4-yl)-benzene-1,4-diamine;
(1H-Indazol-5-yl)-(6-phenyl-thieno[3,2-d]pyrimidin-4-yl)-amine;
[5-(4-Chloro-phenyl)-2H-pyrazol-3-yl]-(6-phenyl-thieno[3,2-d]pyrimidin-4-yl)-amine;
Benzothiazol-6-yl-(6-phenyl-thieno[3,2-d]pyrimidin-4-yl)-amine;
4-(6-Phenyl-thieno[3,2-d]pyrimidin-4-ylamino)-benzamide;
4-Methyl-N-[4-(6-phenyl-thieno[3,2-d]pyrimidin-4-ylamino)-phenyl]-benzenesulfonamide;
N-Phenyl-Nxe2x80x2-(6-phenyl-thieno[3,2-d]pyrimidin-4-yl)-benzene-1,4-diamine;
(6-Phenyl-thieno[3,2-d]pyrimidin-4-yl)-(2H-pyrazol-3-yl)-amine;
(1 H-Indazol-6-yl)-(6-phenyl-thieno[3,2-d]pyrimidin-4-yl)-amine;
N,N-Dimethyl-Nxe2x80x2-(6-phenyl-thieno[3,2-d]pyrimidin-4-yl)-benzene-1,4-diamine;
(2,3-Dimethyl-1H-indol-5-yl)-(6-phenyl-thieno[3,2-d]pyrimidin-4-yl)-amine;
N-Ethyl-Nxe2x80x2-(6-phenyl-thieno[3,2-d]pyrimidin-4-yl)-benzene-1,4-diamine;
[4-(1H-Indol-5-ylamino)-thieno[3,2-d]pyrimidin-6-yl]-phenyl-methanone;
(1H-Indol-5-yl)-(6-p-tolyl-thieno[3,2-d]pyrimidin-4-yl)-amine;
(5-Furan-2-yl-2H-pyrazol-3-yl)-thieno[3,2-d]pyrimidin-4-yl-amine;
Thieno[3,2-d]pyrimidin-4-yl-(5-thiophen-2-yl-2H-pyrazol-3-yl)-amine;
[5-(4-Chloro-phenyl)-2H-pyrazol-3-yl]-thieno[3,2-d]pyrimidin-4-yl-amine;
(2H-Pyrazol-3-yl)-thieno[3,2-d]pyrimidin-4-yl-amine;
Thieno[3,2-d]pyrimidin-4-yl-(5-p-tolyl-2H-pyrazol-3-yl)-amine;
4-[4-(1H-Indol-5-ylamino)-thieno[3,2-d]pyrimidin-6-yl]-benzaldehyde;
[6-(4-Chloro-phenyl)-thieno[3,2-d]pyrimidin-4-yl]-(1H-indol-5-yl)-amine;
[6-(4-Fluoro-phenyl)-thieno[3,2-d]pyrimidin-4-yl]-(1H-indol-5-yl)-amine;
(1H-Indol-5-yl)(6-thiophen-3-yl-thieno[3,2-d]pyrimidin-4-yl)-amine;
2-[3-(4-Chloro-phenyl)-5-(thieno[3,2-d]pyrimidin-4-ylamino)-pyrazol-1-yl]-ethanol;
(1H-Indol-5-yl)-[6-(4-trifluoromethyl-phenyl)-thieno[3,2-d]pyrimidin-4-yl]-amine;
(1H-Indol-5-yl)-[6-(4-methylsulfanyl-phenyl)-thieno[3,2-d]pyrimidin-4-yl]-amine;
(1H-Indol-5-yl)-[6-(3-nitro-phenyl)-thieno[3,2-d]pyrimidin-4-yl]-amine;,
[6-(3-Chloro-4-fluoro-phenyl)-thieno[3,2-d]pyrimidin-4-yl]-(1H-indol-5-yl)-amine;
[5-(4-Methoxy-phenyl)-2H-pyrazol-3-yl]-thieno[3,2-d]pyrimidin-4-yl-amine;
4-[5-(Thieno[3,2-d]pyrimidin-4-ylamino)-1H-pyrazol-3-yl]-benzoic acid methyl ester;
(5-Methyl-2H-pyrazol-3-yl)-thieno[3,2-d]pyrimidin-4-yl-amine;
5-(6-Phenyl-thieno[3,2-d]pyrimidin-4-ylamino)-1H-indole-2-carboxylic acid ethyl ester;
(6-Benzofuran-2-yl-thieno[3,2-d]pyrimidin-4-yl)-(1H-indol-5-yl)-amine;
Thieno[3,2-d]pyrimidin-4-yl-(5-m-tolyl-2H-pyrazol-3-yl)-amine;
[5-(3-Chloro-phenyl)-2H-pyrazol-3-yl]-thieno[3,2-d]pyrimidin-4-yl-amine;
[6-(4-Ethyl-phenyl)-thieno[3,2-d]pyrimidin-4-yl]-(1H-indol-5-yl)-amine;
4-[4-(1H-Indol-5-ylamino)-thieno[3,2-d]pyrimidin-6-yl]-benzoic acid methyl ester;
4-[5-(Thieno[3,2-d]pyrimidin-4-ylamino)-1H-pyrazol-3-yl]-benzoic acid;
(1H-Indol-5-yl)-(6-thiophen-2-yl-thieno[3,2-d]pyrimidin-4-yl)-amine;
[5-(2-Chloro-phenyl)-2H-pyrazol-3yl]thieno[3,2d]pyrimidin-4yl-amine:
(1H-Indol-5-yl)-(6-pyridin-3-yl-thieno[3,2-d]pyrimidin-4-yl)-amine;
(1H-Indol-5-yl)-[6-(3-methoxy-phenyl)-thieno[3,2-d]pyrimidin-4-yl]-amine;
{4-[4-(1H-Indol-5-ylamino)-thieno[3,2-d]pyrimidin-6-yl]-phenyl}-methanol;
[6-(3,4-Dimethoxy-phenyl)-thieno[3,2-d]pyrimidin-4-yl]-(1H-indol-5-yl)-amine;
[6-(4-Dimethylamino-phenyl)-thieno[3,2-d]pyrimidin-4-yl]-(1H-indol-5-yl)-amine;
[4-(1H-Indol-5-ylamino)-thieno[3,2-d]pyrimidin-6-yl]-phenyl-methanol;
4-(1H-Indol-5-ylamino)-thieno[3,2-d]pyrimidine-6-carboxylic acid (2-dimethylamino-ethyl)-amide;
(1H-Indol-5-yl)-[6-(4-trifluoromethoxy-phenyl)-thieno[3,2-d]pyrimidin-4-yl]-amine;
(1H-Indol-5-yl)-[6-(2-methoxy-phenyl)-thieno[3,2-d]pyrimidin-4-yl]-amine;
4-[4-(1H-Indol-5-ylamino)-thieno[3,2-d]pyrimidin-6-yl]-phenol;
[6-(5-Diethoxymethyl-thiophen-2-yl)-thieno[3,2-d]pyrimidin-4-yl]-(1H-indol-5-yl)-amine;
4-(1H-Indol-5-ylamino)-thieno[3,2-d]pyrimidine-6-carboxylic acid (2-methoxy-ethyl)-amide;
N-{4-[4-(1H-Indol-5-ylamino)-thieno[3,2-d]pyrimidin-6-yl]-benzyl}-Nxe2x80x2,Nxe2x80x2-dimethyl-ethane-1,2-diamine;
(1H-Indol-5-yl)-(6-{4-[(2-methoxy-ethylamino)-methyl]-phenyl}-thieno[3,2-d]pyrimidin-4-yl)-amine;
(1H-Indol-5-yl)-{6-[2-(4-methyl-piperazin-1-yl)-phenyl]-thieno[3,2-d]pyrimidin-4-yl}-amine;
4-(1H-Indol-5-ylamino)-thieno[3,2-d]pyrimidine-6-carboxylic acid propylamide;
2-{4-[4-(1H-Indol-5-ylamino)-thieno[3,2-d]pyrimidin-6-yl]-benzylamino}-ethanol;
[6-(2,4-Dimethoxy-phenyl)-thieno[3,2-d]pyrimidin-4-yl]-(1H-indol-5-yl)-amine;
[6-(4-Diethylamino-phenyl)-thieno[3,2-d]pyrimidin-4-yl]-(1H-indol(-5-yl)-amine;
[6-(4-Ethoxy-phenyl)-thieno[3,2-d]pyrimidin-4-yl]-(1H-indol-5-yl)-amine;
3-{4-[4-(1H-Indol-5-ylamino)-thieno[3,2-d]pyrimidin-6-yl]-benzylamino}-propane-1,2-diol;
(1H-Indol-5-yl)-[6-(4-propylaminomethyl-phenyl)-thieno[3,2-d]pyrimidin-4-yl]-amine;
(1H-(Indol-5-yl)-(6-{4-[(3-methoxy-propylamino)-methyl]-phenyl}-thieno[3,2-d]pyrimidin-4-yl)-amine;
[6-(3-Fluoro-4-methoxy-phenyl)-thieno[3,2-d]pyrimidin-4-yl]-(1H-indol-5-yl)-amine;
(1H-Indol-5-yl)-[6-(3-methylsulfanyl-phenyl)-thieno[3,2-d]pyrimidin-4-yl]-amine;
(1H-Indol-5-yl)-[6-(5-methyl-thiophen-2-yl)-thieno[3,2-d]pyrimidin-4-yl]-amine;
(1H-Indol-5-yl)-(6-{4-[(2-piperazin-1-yl-ethyl amino)-methyl]-phenyl}-thieno[3,2-d]pyrimidin-4-yl)-amine;
(6-Benzo[1,3]dioxol-5-yl-thieno[3,2-d]pyrimidin-4-yl)-(1H-indol-5-yl)-amine;
{6-[4-(1-Ethoxy-ethoxy)-phenyl]-thieno[3,2-d]pyrimidin-4-yl}-(1H-indol-5-yl)-amine;
(1H-Indol-5-yl)-{6-[4-(2-pyrrolidin-1-yl-ethoxy)-phenyl]-thieno[3,2-d]pyrimidin-4-yl}-amine;
(1H-Indol-5-yl)-(6-[4-(2-methoxy-ethoxy)-phenyl]-thieno[3,2-d]pyrimidin-4-yl)-amine;
(1H-indol-5-yl)-(6-{4-[(2-morpholin-4-yl-ethylamino)-methyl]-phenyl}-thieno[3,2-d]pyrimidin-4-yl)-amine;
{6-[4-(2-Dimethylamino-ethoxy)-phenyl]-thieno[3,2-d]pyrimidin-4-yl}-(1H-indol-5-yl)-amine;
(1H-indol-5-yl)-[6-(4-methylaminomethyl-phenyl)-thieno[3,2-d]pyrimidin-4-yl]-amine;
5-(6-Phenyl-thieno[3,2-d]pyrimidin-4-ylamino)-1 ,3-dihydro-indol-2-one;
(1H-Benzotriazol-5-yl)-(6-phenyl-thieno[3,2-d]pyrimidin-4-yl)-amine;
(6-Phenyl-thieno[3,2-d]pyrimidin-4-yl)-[4-(2H-tetrazol-5-yl)-phenyl]-amine;
N-{4-[4-(1H-Indol-5-ylamino)-thieno[3,2-d]pyrimidin-6-yl]-benzyl}-Nxe2x80x2-methyl-ethane-1,2-diamine;
(1-Benzenesulfonyl-1H-indol-5-yl)-(6-phenyl-thieno[3,2-d]pyrimidin-4-yl)-amine;
3-{4-[4-(1H-Indol-5-ylamino)-thieno[3,2-d]pyrimidin-6-yl]-benzylamino}-propan-1-ol;
(1H-indol-5-yl)-{6-[4-(4-methyl-piperazin-1-ylmethyl)-phenyl)-thieno[3,2-d]pyrimidin-4-yl}-amine;
2-{4-[4-(1H-Indol-5-ylamino)-thieno[3,2-d]pyrimidin-6-yl]-benzylamino}-propane-1,3-diol;
2-((2-Hydroxy-ethyl)-{4-(4-(1H-indol-5-ylamino)-thieno[3,2-d]pyrimidin-6-yl]-benzyl}-amino)-ethanol;
{5-[4-(1H-Indol-5-ylamino)-thieno[3,2-d]pyrimidin-6-yl]-thiophen-2-yl}-methanol;
2-(2-{4-[4-(1H-Indol-5-ylamino)-thieno[3,2-d]pyrimidin-6-yl]-benzylamino}-ethoxy)-ethanol;
2-(2-{4-[4-(1H-Indol-5-ylamino)-thieno[3,2-d]pyrimidin-6-yl]-benzylamino}-ethylamino)-ethanol;
[6-(4-{[2-(1H-Imidazol-4-yl)-ethylamino]-methyl}-phenyl)-thieno[3,2-d]pyrimidin-4-yl]-(1H-indol-5-yl)-amine;
(1H-Indol-5-yl)-{6-[4-(2-morpholin-4-yl-ethoxy)-phenyl]-thieno[3,2-d]pyrimidin-4-yl}-amine;
2-{4-[4-(1H-Indol-5-ylamino)-thieno[3,2-d]pyrimidin-6-yl]-phenoxy}-ethanol;
[4-(2-Ethyl-oxazol-5-yl)-phenyl]-(6-phenyl-thieno[3,2-d]pyrimidin-4-yl)-amine;
N-(2-Methoxy-phenyl)-Nxe2x80x2-(6-phenyl-thieno[3,2-d]pyrimidin-4-yl)-benzene-1,4-diamine;
N-(4-Methoxy-phenyl)-Nxe2x80x2-(6-phenyl-thieno[3,2-d]pyrimidin-4-yl)-benzene-1,4-diamine;
5-[4-(1H-Indol-5-ylamino)-thieno[3,2-d]pyrimidin-6-yl]-thiophene-2-carbaldehyde;
[5-(6-Phenyl-thieno[3,2-d]pyrimidin-4-ylamino)-1H-indol-2-yl]-methanol;
(2-Phenyl-1H-indol-3-yl)-(6-phenyl-thieno[3,2-d]pyrimidin-4-yl)-amine;
(9H-Carbazol-3-yl)-(6-phenyl-thieno[3,2-d]pyrimidin-4-yl)-amine;
(2-Methyl-1H-indol-5-yl)-(6-phenyl-thieno[3,2-d]pyrimidin-4-yl)-amine;
(1-Phenyl-ethyl)-(6-phenyl-thieno[3,2-d]pyrimidin-4-yl)-amine;
(1H-Indol-5-yl)-[6-(4-{[(thiophen-2-ylmethyl)-amino]-methyl}-phenyl)-thieno[3,2-d]pyrimidin-4-yl]-amine;
3-{4-[4-(1H-Indol-5-ylamino)-thieno[3,2-d]pyrimidin-6-yl]-benzylamino}-propionic acid methyl ester;
[6-(4-{[(Furan-2-ylmethyl)-amino]-methyl}-phenyl)-thieno[3,2-d]pyrimidin-4-yl]-(1H-indol-5-yl)-amine;
1-(3-{4-[4-(1H-Indol-5-ylamino)-thieno[3,2-d]pyrimidin-6-yl]-benzylamino}-propyl)-pyrrolidin-2-one;
N-{4-[4-(1H-Indol-5-ylamino)-thieno[3,2-d]pyrimidin-6-yl]-benzyl}-Nxe2x80x2,Nxe2x80x2-dimethyl-propane-1,3-diamine;
(1H-Indol-5-yl)-[6-(4-morpholin-4-ylmethyl-phenyl)-thieno[3,2-d]pyrimidin-4-yl)-amine;
(2-{4-[4-(1H-Indol-5-ylamino)-thieno[3,2-d]pyrimidin-6-yl]-benzylamino}-acetylamino)-acetic acid ethyl ester;
1-(4-{4-[4-(1H-indol-5-ylamino)-thieno[3,2-d]pyrimidin-6-yl]-benzyl}-piperazin-1-yl)-ethanone;
(6-{4-[(2,2-Diphenyl-ethylamino)-methyl]-phenyl}-thieno[3,2-d]pyrimidin-4-yl)-(1H-indol-5-yl)-amine;
(1H-indol-5-yl)-{6-[4-(2-methoxymethyl-pyrrolidin-1-ylmethyl)-phenyl]-thieno[3,2-d]pyrimidin-4-yl}-amine;
N-(2-{4-[4-(1H-Indol-5-ylamino)-thieno[3,2-d]pyrimidin-6-yl]-benzylamino}-ethyl)-acetamide;
[6-(4-Cyclopropylaminomethyl-phenyl)-thieno[3,2-d]pyrimidin-4-yl]-(1H-indol-5-yl)-amine;
2-{4-[4-(1H-Indol-5-ylamino)-thieno[3,2-d]pyrimidin-6-yl]-benzylamino}-butan-1-ol;
2-({5-[4-(1H-Indol-5-ylamino)-thieno[3,2-d]pyrimidin-6-yl]-thiophen-2-ylmethyl}-amino)-ethanol;
(1H-Indol-5-yl)-(6-{4-[(2-pyrrolidin-1-yl-ethylamino)-methyl]-phenyl}-thieno[3,2-d]pyrimidin-4-yl)-amine;
(1H-Indol-5-yl)-(2-thiophen-2-yl-thieno[3,2-b]pyridin-7-yl)-amine;
{6-[4-(Benzylamino-methyl)-phenyl]-thieno[3,2-d]pyrimidin-4-yl}-(1H-indol-5-yl)-amine;
1-{4-[4-(1H-Indol-5-ylamino)-thieno[3,2-d]pyrimidin-6-yl]-benzyl}-piperidine-4-carboxylic acid amide;
(1H-Indol-5-yl)-{6-[4-(pyrrolidin-3-ylaminomethyl)-phenyl]-thieno[3,2-d]pyrimidin-4yl}-amine;
4-[7-(1H-Indol-5-ylamino)-thieno[3,2-b]pyridin-2-yl]-benzaldehyde;
(6-{4-[(3-Imidazol-1-yl-propylamino)-methyl]-phenyl}-thieno[3,2-d]pyrimidin-4-yl)-(1H-indol-5-yl)-amine;
N-{4-[4-(1H-Indol-5-ylamino)-thieno[3,2-d]pyrimidin-6-yl]-benzyl}-Nxe2x80x2,Nxe2x80x2-dimethyl-hexane-1,6-diamine;
(1-Allyl-1H-indol-5-yl)-(6-phenyl-thieno[3,2-d]pyrimidin-4-yl)-amine;
(1-Methyl-1H-indol-5-yl)-(6-phenyl-thieno[3,2-d]pyrimidin-4-yl)-amine;
(1H-Indol-5-yl)-{6-[4-(4-phenyl-piperazin-1-ylmethyl)-phenyl]-thieno[3,2-d]pyrimidin-4-yl}-amine;
N-{5-[4-(1H-indol-5-ylamino)-thieno[3,2-d]pyrimidin-6-yl]-thiophen-2-ylmethyl}-Nxe2x80x2,Nxe2x80x2-dimethyl-ethane-1,2-diamine;
N-{5-[4-(1H-indol-5-ylamino)-thieno[3,2-d]pyrimidin-6-yl]-thiophen-2-ylmethyl}-Nxe2x80x2-methyl-ethane-1,2-diamine;
(1H-Indol-5-yl)-(6-{5-[(2-methoxy-ethylamino)-methyl]-thiophen-2-yl}-thieno[3,2-d]pyrimidin-4-yl)-amine;
2-Amino-3-(3-{4-[4-(1H-indol-5-ylamino)-thieno[3,2-d]pyrimidin-6-yl]-benzyl}-3H-imidazol-4-yl)-propionic acid methyl ester;
3-{4-[4-(1H-Indol-5-ylamino)-thieno[3,2-d]pyrimidin-6-yl]-benzylamino}-2,2-dimethyl-propan-1-ol;
4-[7-(1H-Indol-5-ylamino)-thieno[3,2-b]pyridin-2-yl]-phenol;
(9-Ethyl-9H-carbazol-3-yl)-(6-phenyl-thieno[3,2-d]pyrimidin-4-yl)-amine;
[1-(2-Diethylamino-ethyl)-1H-indol-5-yl]-(6-phenyl-thieno[3,2-d]pyrimidin-4-yl)-amine;
[1-(3-Diethylamino-propyl)-1H-indol-5-yl]-(6-phenyl-thieno[3,2-d]pyrimidin-4-yl)-amine;
(2-Bromo-thieno[3,2-b]pyridin-7-yl)-(1H-indol-5-yl)-amine;
[6-(4-Aminomethyl-phenyl)-thieno[3,2-d]pyrimidin-4-yl]-(1H-indol-5-yl)-amine;
3-Hydroxy-2-{4-[4-(1H-indol-5-ylamino)-thieno[3,2-d]pyrimidin-6-yl]-benzylamino}-propionic acid methyl ester;
Furan-2-yl-(4-{4-[4-(1H-indol-5-ylamino)-thieno[3,2-d]pyrimidin-6-yl]-benzyl}-piperazin-1-yl)-methanone;
(1H-indol-5-yl)-[2-(4-methylsulfanyl-phenyl)-thieno[3,2-b]pyridin-7-yl]-amine;
(6-(4-Dimethylaminomethyl-phenyl)-thieno[3,2-d]pyrimidin-4-yl]-(1H-indol-5-yl)-amine;
2-({4-[4-(1H-Indol-5-ylamino)-thieno[3,2-d]pyrimidin-6-yl]-benzyl}-methyl-amino)-ethanol;
(1-{4-[4-(1H-Indol-5-ylamino)-thieno[3,2-d]pyrimidin-6-yl]-benzyl}-pyrrolidin-2-yl)-methanol;
2-[2-(4-{4-[4-(1H-Indol-5-ylamino)-thieno[3,2-d]pyrimidin-6-yl]-benzyl}-piperazin-1-yl)-ethoxy]-ethanol;
[2-(4-Fluoro-phenyl)-thieno[3,2-b]pyridin-7-yl]-(1H-indol-5-yl)-amine;
4-[4-(1H-Indol-5-ylamino)-thieno[3,2-d]pyrimidin-6-yl]-benzoic acid;
(1H-Indol-5-yl)-thieno[3,2-b]pyridin-7-yl-amine;
2-{4-[7-(1H-Indol-5-ylamino)-thieno[3,2-b]pyridin-2-yl]-phenoxy}-ethanol;
(1H-Indol-5-yl)-(2-methyl-6-phenyl-thieno[3,2-d]pyrimidin-4-yl)-amine;
N-(4-Methoxy-phenyl)-Nxe2x80x2-(2-methyl-6-phenyl-thieno[3,2-d]pyrimidin-4-yl)-benzene-1,4-diamine;
N-(2-Benzyloxy-ethyl)-Nxe2x80x2-(6-phenyl-thieno[3,2-d]pyrimidin-4-yl) -benzene-1,4-diamine;
5-(6-Phenyl-thieno[3,2-d]pyrimidin-4-ylamino)-1H-indole-3-carbaldehyde;
[2-(4-Dimethylamino-phenyl)-thieno[3,2-b]pyridin-7-yl]-(1H-indol-5-yl)-amine;
4-[7-(1H-Indol-5-ylamino)-thieno[3,2-b]pyridin-2-yl]-benzoic acid methyl ester;
(1H-Indol-5-yl)-(2-thiophen-3-yl-thieno[3,2-b]pyridin-7-yl)-amine;
(1H-Indol-5-yl)-(2-{4-[(2-methoxy-ethylamino)-methyl]-phenyl}-thieno[3,2-b]pyridin-7-yl)-amine;
Furan-2-yl-(4-{4-[7-(1H-indol-5-ylamino)-thieno[3,2-b]pyridin-2-yl]-benzyl}-piperazin-1-yl)-methanone;
(3-Bromo-1H-indol-5-yl)-(6-phenyl-thieno[3,2-d]pyrimidin-4-yl)-amine;
N-(1H-Indol-3-ylmethyl)-Nxe2x80x2-(6-phenyl-thieno[3,2-d]pyrimidin-4-yl)-benzene-1,4-diamine;
N-(6-Bromo-thieno[3,2-d]pyrimidin-4-yl)-Nxe2x80x2-(4-methoxy-phenyl)-benzene-1,4-diamine;
N-(4-Methoxy-phenyl)-Nxe2x80x2-[6-(2-nitro-phenyl)-thieno[3,2-d]pyrimidin-4-yl]-benzene-1,4-diamine;
N-(4-Methoxy-phenyl)-Nxe2x80x2-[6-(4-methoxy-phenyl)-thieno[3,2-d]pyrimidin-4-yl]-benzene-1,4-diamine;
N-(4-Methoxy-phenyl)-Nxe2x80x2-[6-(6-methoxy-pyridin-3-yl 3-thieno[3,2-d]pyrimidin-4-yl]-benzene-1,4-diamine;
N-(4-Methoxy-phenyl)-Nxe2x80x2-(6-thiophen-2-yl-thieno[3,2-d]pyrimidin-4-yl)-benzene-1,4-diamine;
(1H-Indol-5-yl)-[6-(4-thiomorpholin-4-ylmethyl-phenyl)-thieno[3,2-d]pyrimidin-4-yl]-amine;
2-(2-{4-[7-(1H-Indol-5-ylamino)-thieno[3,2-b]pyridin-2-yl]-benzylamino}-ethoxy)-ethanol;
2-{4-[7-(1H-Indol-5-ylamino)-thieno[3,2-b]pyridin-2-yl]-benzylamino}-ethanol;
N-{4-[7-(1H-indol-5-ylamino)-thieno[3,2-b]pyridin-2-yl]-benzyl}-Nxe2x80x2,Nxe2x80x2-dimethyl-hexane-1,6-diamine;
2-({4-[7-(1H-indol-5-ylamino)-thieno[3,2-b]pyridin-2-yl]-benzyl}-methyl-amino)-ethanol
(1H-Indol-5-yl)-(2-{4-[(2-piperazin-1-yl-ethylamino)-methyl]-phenyl}-thieno[3,2-b]pyridin-7-yl)-amine;
(2-{4-[(3-Imidazol-1-yl-propylamino)-methyl]-phenyl}-thieno[3,2-b]pyridin-7-yl)-(1H-indol-5-yl)-amine;
2-((2-Hydroxy-ethyl)-{4-[7-(1H-indol-5-ylamino)-thieno[3,2-b]pyridin-2-yl]-benzyl}-amino)-ethanol;
[2-(4-Dimethylaminomethyl-phenyl)-thieno[3,2-b]pyridin-7-yl]-(1H-indol-5-yl)-amine;
N-{4-[7-(1H-Indol-5-ylamino)-thieno[3,2-b]pyridin-2-yl]-benzyl}-Nxe2x80x2,Nxe2x80x2-dimethyl-ethane-1,2-diamine;
(1-{4-[7-(1H-Indol-5-ylamino)-thieno[3,2-b]pyridin-2-yl]-benzyl}-pyrrolidin-2-yl)-methanol;
2-(4-{4-[7-(1H-Indol-5-ylamino)-thieno[3,2-b]pyridin-2-yl]-benzyl}-piperazin-1-yl)-ethanol;
(1H-Indol-5-yl)-{2-[4-(2-morpholin-4-yl-ethoxy)-phenyl]-thieno[3,2-b]pyridin-7-yl}-amine;
1-{4-[7-(1H-indol-5-ylamino)-thieno[3,2-b]pyridin-2-yl]-benzyl}-piperidine-4-carboxylic acid amide;
{4-[7-(1H-Indol-5-ylamino)-thieno[3,2-b]pyridin-2-yl]-phenyl}-methanol;
6-(6-Phenyl-thieno[3,2-d]pyrimidin-4-ylamino)-benzothiazole-2-thiol;
2-{4-[7-(1H-Indol-5-ylamino)-thieno[3,2-b]pyridin-2-yl]-benzylamino}-butan-1-ol;
N-{4-[7-(1H-Indol-5-ylamino)-thieno[3,2-b]pyridin-2-yl]-benzyl}-Nxe2x80x2-methyl-ethane-1,2-diamine;
(1H-Indol-5-yl)-[2-(4-morpholin-4-ylmethyl-phenyl)-thieno[3,2-b]pyridin-7-yl]-amine;
3-{4-[7-(1H-Indol-5-ylamino)-thieno[3,2-b]pyridin-2-yl]-benzylamino}-propan-1-ol;
1-(3-{4-[7-(1H-Indol-5-ylamino)-thieno[3,2-b]pyridin-2-yl]-benzylamino}-propyl)-pyrrolidin-2-one;
(3-Methyl-1H-indol-5-yl)-(6-phenyl-thieno[3,2-d]pyrimidin-4-yl)-amine;
(1H-indol-5-yl)-{2-[4-(2-methoxy-ethoxy)-phenyl]-thieno[3,2-b]pyridin-7-yl}-amine;
2-(2-{4-[7-(1H-Indol-5-ylamino)-thieno[3,2-b]pyridin-2-yl]-benzylamino}-ethylamino)-ethanol;
3-{4-[7-(1H-indol-5-ylamino)-thieno[3,2-b]pyridin-2-yl]-benzylamino}-2,2-dimethyl-propan-1-ol;
3-{4-[7-(1H-Indol-5-ylamino)-thieno[3,2-b]pyridin-2-yl]-benzylamino}-propane-1,2-diol;
[2-(4-{[2-(1H-Imidazol-4-yl)-ethylamino]-methyl}-phenyl)-thieno[3,2-b]pyridin-7-yl]-(1H-indol-5-yl)-amine;
N-(2-{4-[7-(1H-indol-5-ylamino)-thieno[3,2-b]pyridin-2-yl]-benzylamino}-ethyl)-acetamide;
2-{4-[7-(1H-indol-5-ylamino)-thieno[3,2-b]pyridin-2-yl]-benzylamino}-acetamide;
2-{4-[7-(1H-Indol-5-ylamino)-thieno[3,2-b]pyridin-2-yl]-benzylamino}-propane-1,3-diol;
N-(4-Methoxy-phenyl)-Nxe2x80x2-[2-(3-nitro-phenyl)-thieno[3,2-b]pyridin-7-yl]-benzene-1,4-diamine;
(7-Methoxy-1H-indol-5-yl)-(2-phenyl-thieno[3,2-b]pyridin-7-yl)-amine;
(1H-indol-5-yl)-[2-(4-methylaminomethyl-phenyl)-thieno[3,2-b]pyridin-7-yl]-amine;
N-{4-[7-(1H-Indol-5-ylamino)-thieno[3,2-b]pyridin-2-yl]-benzyl}-ethane-1,2-diamine;
{4-[7-(1H-Indol-5-ylamino)-thieno[3,2-b]pyridin-2-yl]-benzylamino}-acetic acid methyl ester;
N-{4-[7-(1H-Indol-5-ylamino)-thieno[3,2-b]pyridin-2-yl]-benzyl}-Nxe2x80x2,Nxe2x80x2-dimethyl-propane-1,3-diamine;
N-(4-Methoxy-phenyl)-Nxe2x80x2-thieno[3,2-d]pyrimidin-4-yl-benzene-1,4-diamine;
(1H-Indol-5-yl)-(2-pyridin-2-yl-thieno[3,2-b]pyridin-7-yl)-amine;
(1H-Indol-5-yl)-(2-{4-[(2-morpholin-4-yl-ethylamino)-methyl]-phenyl}-thieno[3,2-b]pyridin-7-yl)-amine;
(1H-Indol-5-yl)-{2-[4-(pyrrolidin-3-ylaminomethyl)-phenyl]-thieno[3,2-b]pyridin-7-yl}-amine;
1-(4-{4-[7-(1H-Indol-5-ylamino)-thieno[3,2-b]pyridin-2-yl]-benzyl}-piperazin-1-yl)-ethanone;
1-{4-[7-(1H-Indol-5-ylamino)-thieno[3,2-b]pyridin-2-yl]-benzyl}-pyrrolidine-2-carboxylic acid amide;
N-(4-Methoxy-phenyl)-Nxe2x80x2-[2-(6-methoxy-pyridin-3-yl)-thieno[3,2-b]pyridin-7-yl]-benzene-1,4-diamine;
(1H-indol-5-yl)-(2-pyridin-3-yl-thieno[3,2-b]pyridin-7-yl)-amine;
N-(2-Methoxy-phenyl)-Nxe2x80x2-(6-phenyl-thieno[3,2-d]pyrimidin-4-yl)-benzene-1,4-diamine;
N-(6-Phenyl-thieno[3,2-d]pyrimidin-4-yl)-Nxe2x80x2-o-tolyl-benzene-1,4-diamine;
N-(6-Phenyl-thieno[3,2-d]pyrimidin-4-yl)-Nxe2x80x2-p-tolyl-benzene-1,4-diamine;
N-(3,4-Dimethoxy-phenyl)-Nxe2x80x2-(6-phenyl-thieno[3,2-d]pyrimidin-4-yl)-benzene-1,4-diamine;
N-(6-Phenyl-thieno[3,2-d]pyrimidin-4-yl)-Nxe2x80x2-(3,4,5-trimethoxy-phenyl)-benzene-1,4-diamine;
N-(6-Phenyl-thieno[3,2-d]pyrimidin-4-yl)-Nxe2x80x2-m-tolyl-benzene-1,4-diamine;
N-(4-Chloro-phenyl)-Nxe2x80x2-(6-phenyl-thieno[3,2-d)benzene-1,4diamine;
4-[7-(1H-indol-5-ylamino)-thieno[3,2-b]pyridin-2-yl]-but-3-yn-1-ol;
(1H-indol-5-yl)-[6-(6-methoxy-pyridin-3-yl)-thieno[3,2d]pyrimidin-4-yl]-amine;
N-(4dimethylamino-phenyl)-Nxe2x80x2-(6-phenyl-thieno[3,2-d]pyrimidin-4-yl)benzene-1,4-diamine;
N-(3-Methoxy-phenyl)-Nxe2x80x2-(6-phenyl-thieno[3,2-d]pyrimidin-4-yl)-benzene-1,4-diamine;
(1,3-Dibromo-1H-indol-5-yl)-(6-phenyl-thieno[3,2-d]pyrimidin-4-yl)-amine;
N-(4-Methoxy-phenyl)-Nxe2x80x2-(2-thiophen-2-yl-thieno[3,2-b]pyridin-7-yl)-benzene-1,4-diamine;
(6-Chloro-1H-indol-5-yl)-(6-phenyl-thieno[3,2-d]pyrimidin-4-yl)-amine;
[5-(6-Phenyl-thieno[3,2-d]pyrimidin-4-ylamino)-1H-indol-3-yl]-methanol;
N-(2-Hydroxy-ethyl)-4-[7-(1H-indol-5-ylamino)-thieno[3,2-b]pyridin-2-yl]-benzamide;
N-(3-Imidazol-1-yl-propyl)-4-[7-(1H-indol-5-ylamino)-thieno[3,2-b]pyridin-2-yl]-benzamide;
3-[4-(4-{4-[7-(1H-Indol-5-ylamino)-thieno[3,2-b]pyridin-2-yl]-benzylamino}-butyl)-piperazin-1-yl]-propan-1-ol;
(1H-Indol-5-yl)-[2-(4-{[4-(4-methyl-piperazin-1-yl)-butylamino]-methyl}-phenyl)-thieno[3,2-b]pyridin-7-yl]-amine;
2-[4-(4-{4-[7-(1H-Indol-5-ylamino)-thieno[3,2-b]pyridin-2-yl]-benzylamino}-butyl)-piperazin-1-yl]-ethanol;
1-Imidazol-1-yl-3-{4-[7-(1H-indol-5-ylamino)-thieno[3,2-b]pyridin-2-yl]-benzylamino}-propan-2-ol;
5-{4-[7-(1H-Indol-5-ylamino)-thieno[3,2-b]pyridin-2-yl]-benzylamino}-pentan-1ol;
2-[2-(4-{4-[7-(1H-Indol-5-ylamino)-thieno[3,2-b]pyridin-2-yl]-benzyl}-piperazin-1yl)-ethoxy]-ethanol;
(1H-indol-5-yl)-(2-{4-[(2-methylsulfanyl-ethylamino)-methyl]-phenyl}-thieno[3,2-b]pyridin-7-yl)-amine;
2-[(2-Hydroxy-ethyl)-(3-{4-[7-(1H-indol-5-ylamino)-thieno[3,2-b]pyridin-2yl]-benzylamino}-propyl)-amino]-ethanol;
N-(2-Amino-ethyl)-Nxe2x80x2-{4-[7-(1H-indol-5-ylamino)-thieno[3,2-b]pyridin-2-yl]-benzyl}-ethane-1,2-diamine;
2-(3-{4-[7-(1H-Indol-5-ylamino)-thieno[3,2-b]pyridin-2-yl]-benzylamino}-propylamino)-ethanol;
N-{4-[7-(1H-Indol-5-ylamino)-thieno[3,2-b]pyridin-2-yl]-benzyl}-hexane-1,6-diamine
(2-Methyl-1H-indol-5-yl)-[2-(4-morpholin-4-ylmethyl-phenyl)-thieno[3,2-b]pyridin-7-yl]amine;
(6-Phenyl-thieno[3,2-d]pyrimidin-4-yl)-{3-[(3-pyrazol-1-yl-propylamino)-methyl]-1H-indol-5-yl}-amine;
{[5-(6-Phenyl-thieno[3,2-d]pyrimidin-4-ylamino)-1H-indol-3-ylmethyl]-amino}-acetic acid methyl ester;
2-{[5-(6-Phenyl-thieno[3,2-d]pyrimidin-4-ylamino)-1H-indol-3-ylmethyl]-amino}-ethanol;
2-{4-[7-(2-Methyl-1H-indol-5-ylamino)-thieno[3,2-b]pyridin-2-yl]-benzylamino}-ethanol;
(1H-Indol-5-yl)-[2-(6-methoxy-pyridin-3-yl)-thieno[3,2-b]pyridin-7-yl]-amine;
{5-[7-(1H-Indol-5-ylamino)-thieno[3,2-b]pyridin-2-yl]-pyridin-2-yl}-methanol;
N,N-Dimethyl-Nxe2x80x2-{4-[7-(2-methyl-1H-indol-5-ylamino)-thieno[3,2-b]pyridin-2-yl]-benzyl}-propane-1,3-diamine;
5-(6-Phenyl-thieno[3,2-d]pyrimidin-4-ylamino)-1H-indole-3-carbaldehyde oxime;
(3-Methyliminomethyl-1H-indol-5-yl)-(6-phenyl-thieno[3,2-d]pyrimidin-4-yl)-amine;
[3-(2-Nitro-vinyl)-1H-indol-5-yl]-(6-phenyl-thieno[3,2-d]pyrimidin-4-yl)-amine;
4-[4-(6-Phenyl-thieno[3,2-d]pyrimidin-4-ylamino)-phenylamino]-phenol;
5-Methyl-1-[4-(6-phenyl-thieno[3,2-d]pyrimidin-4-ylamino)-phenyl]-1,2-dihydro-pyrazol-3-one;
(2-Methyl-benzothiazol-6-yl)-(6-phenyl-thieno[3,2-d]pyrimidin-4-yl)-amine;
2-[(2-Hydroxy-ethyl)-(3-{4-[7-(2-methyl-1H-indol-5-ylamino)-thieno[3,2-b]pyridin-2-yl]-benzylamino}-propyl)-amino]-ethanol;
2-{4-[7-(2-Methyl-1H-indol-5-ylamino)-thieno[3,2-b]pyridin-2-yl]-benzylamino}-propane-1,3-diol;
3-{4-[7-(2-Methyl-1H-indol-5-ylamino)-thieno[3,2-b]pyridin-2-yl]-benzylamino}-propane-1,2-diol;
1-(3-{4-[7-(2-Methyl-1H-indol-5-ylamino)-thieno[3,2-b]pyridin-2-yl]-benzylamino}-propyl)-pyrrolidin-2-one;
N-(2-Amino-ethyl)-Nxe2x80x2-{4-[7-(2-methyl-1H-indol-5-ylamino)-thieno[3,2-b]pyridin-2-yl]-benzyl}-ethane-1,2-diamine;
2-(2-{4-[7-(2-Methyl-1H-indol-5-ylamino)-thieno[3,2-b]pyridin-2-yl]-benzylamino}-ethylamino)-ethanol;
3-{4-[7-(2-Methyl-1H-indol-5-ylamino)-thieno[3,2-b]pyridin-2-yl]-benzylamino}-propan-1-ol;
1-{4-[7-(2-Methyl-1H-indol-5-ylamino)-thieno[3,2-b]pyridin-2-yl]-benzyl}-piperidine-4-carboxylic acid amide;
2-(2-{4-[7-(2-Methyl-1H-indol-5-ylamino)-thieno[3,2-b]pyridin-2-yl]-benzylamino}-ethoxy)-ethanol;
2-(Methyl-{4-[7-(2-methyl-1H-indol-5-ylamino)-thieno[3,2-b]pyridin-2-yl]-benzyl}-amino)-ethanol;
N-Methyl-Nxe2x80x2-{4-[7-(2-methyl-1H-indol-5-ylamino)-thieno[3,2-b]pyridin-2-yl]-benzyl}-ethane-1,2-diamine;
(1H-Indol-5-yl)-[2-(3-nitro-phenyl)-thieno[3,2-b]pyridin-7-yl]-amine;
N-{4-[7-(2-Methyl-1H-indol-5-ylamino)-thieno[3,2-b]pyridin-2-yl]-benzyl}-ethane-1,2-diamine;
(2-Methyl-1H-indol-5-yl)-(2-{4-[(2-piperazin-1-yl-ethylamino)-methyl]-phenyl}-thieno[3,2-b]pyridin-7-yl)-amine;
N,N-Dimethyl-Nxe2x80x2-{4-[7-(2-methyl-1H-indol-5-ylamino)-thieno[3,2-b]pyridin-2-yl]-benzyl}-ethane-1,2-diamine;
2-{4-[7-(2-Methyl-1H-indol-5-ylamino)-thieno[3,2-b]pyridin-2-yl]-benzylamino}-butan-1-ol;
(2-Methyl-1H-indol-5-yl)-(2-{4-[(2-morpholin-4-yl-ethylamino)-methyl]-phenyl}-thieno[3,2-b]pyridin-7-yl)-amine;
(2-Methyl-1H-indol-5-yl)-{2-[4-(pyrrolidin-3-ylaminomethyl)-phenyl]-thieno[3,2-b]pyridin-7-yl}-amine;
{6-[7-(1H-Indol-5-ylamino)-thieno[3,2-b]pyridin-2-yl]-pyridin-3-yl}-methanol;
{6-[7-(2-Methyl-1H-indol-5-ylamino)-thieno[3,2-b]pyridin-2-yl]-pyridin-3-yl}-methanol
(3-Methylaminomethyl-1H-indol-5-yl)-(6-phenyl-thieno[3,2-d]pyrimidin-4-yl)-amine;
3-[4-(4-{4-[7-(2-Methyl-1H-indol-5-ylamino)-thieno[3,2-b]pyridin-2-yl]-benzylamino}-butyl)-piperazin-1-yl]-propan-1-ol;
2-[4-(4-{4-[7-(2-Methyl-1H-indol-5-ylamino)-thieno[3,2-b]pyridin-2-yl]-benzylamino}-butyl)-piperazin-1-yl]-ethanol;
(2-{4-[(3-Imidazol-1-yl-propylamino)-methyl]-phenyl}-thieno[3,2-b]pyridin-7-yl)-(2-methyl-1H-indol-5-yl)-amine;
1-Imidazol-1-yl-3-{4-[7-(2-methyl-1H-indol-5-ylamino)-thieno[3,2-b]pyridin-2-yl]-benzylamino}-propan-2-ol;
2-[(2-Hydroxy-ethyl)-(4-{4-[7-(2-methyl-1H-indol-5-ylamino)-thieno[3,2-b]pyridin-2-yl]-benzylamino}-butyl)-amino]-ethanol;
N,N-Diethyl-Nxe2x80x2-{4-[7-(2-methyl-1H-indol-5-ylamino)-thieno[3,2-b]pyridin-2-yl]-benzyl}-propane-1,3-diamine;
[2-(3-Amino-phenyl)-thieno[3,2-b]pyridin-7-yl]-(1H-indol-5-yl)-amine;
(2-Methyl-1H-indol-5-yl)-(2-{4-[(3-morpholin-4-yl-propylamino)-methyl]-phenyl}-thieno[3,2-b]pyridin-7-yl)-amine;
[2-(4-Dimethylaminomethyl-phenyl)-thieno[3,2-b]pyridin-7-yl]-(2-methyl-1H-indol-5-yl)-amine;
1-[5-(2-Pyridin-2-yl-thieno[3,2-b]pyridin-7-ylamino)-2,3-dihydro-indol-1-yl]-ethanone;
(2,3-Dihydro-1H-indol-5-yl)-(2-pyridin-2-yl-thieno[3,2-b]pyridin-7-yl)-amine;
(1H-Benzotriazol-5-yl)-(2-pyridin-2-yl-thieno[3,2-b]pyridin-7-yl)-amine;
5-(2-Phenyl-thieno[3,2-b]pyridin-7-ylamino)-1H-indole-3-carbaldehyde;
(1H-Indazol-5-yl)-(2-pyridin-2-yl-thieno[3,2-b]pyridin-7-yl)-amine;
(2-Methyl-1H-indol-5-yl)-(2-pyridin-2-yl-thieno[3,2-b]pyridin-7-yl)-amine;
(1H-Benzoimidazol-5-yl)-(2-phenyl-thieno[3,2-b]pyridin-7-yl)-amine;
5-(2-Pyridin-2-yl-thieno[3,2-b]pyridin-7-ylamino)-1H-indole-2-carboxylic acid dimethylamide;
{5-[7-(2-Methyl-1H-indol-5-ylamino)-thieno[3,2-b]pyridin-2-yl]-pyridin-2-yl}-methanol;
N-(3-Imidazol-1-yl-propyl)-6-[7-(1H-indol-5-ylamino)-thieno[3,2-b]pyridin-2-yl]-nicotinamide;
N-(3-Hydroxy-propyl)-6-[7-(2-methyl-1H-indol-5-ylamino)-thieno[3,2-b]pyridin-2-yl]-nicotinamide;
[2-(5-Amino-pyridin-2-yl)-thieno[3,2-b]pyridin-7-yl]-(2-methyl-1H-indol-5-yl)-amine;
N-[2-(2-Hydroxy-ethoxy)-ethyl]-6-[7-(2-methyl-1H-indol-5-ylamino)-thieno[3,2-b]pyridin-2-yl]-nicotinamide;
(4-Methoxy-2-methyl-phenyl)-(6-phenyl-thieno[3,2-d]pyrimidin-4-yl)-amine;
[4-(4-Chloro-phenoxy)-phenyl]-(6-phenyl-thieno[3,2-d]pyrimidin-4-yl)-amine;
6-(6-Phenyl-thieno[3,2-d]pyrimidin-4-ylamino)-1H-benzo[d][1,3]oxazine-2,4-dione;
2-Diethylaminomethyl-4-(6-phenyl-thieno[3,2-d]pyrimidin-4-ylamino)-phenol;
5-Methyl-1-[4-(6-phenyl-thieno[3,2-d]pyrimidin-4-ylamino)-phenyl]-1,2-dihydro-pyrazol-3-one;
[4-(4,5-Dichloro-imidazol-1-yl)-phenyl]-(6-phenyl-thieno[3,2-d]pyrimidin-4-yl)-amine;
(6-Phenyl-thieno[3,2-d]pyrimidin-4-yl)-[4-(3-trifluoromethyl-pyrazol-1-yl)-phenyl]-amine;
[4-(4-Methyl-piperazin-1-yl)-phenyl]-(6-phenyl-thieno[3,2-d]pyrimidin-4-yl)-amine;
[4-(4-Methyl-piperidin-1-yl)-phenyl]-(6-phenyl-thieno[3,2-d]pyrimidin-4-yl)-amine;
1-[4-(6-Phenyl-thieno[3,2-d]pyrimidin-4-ylamino)-phenyl]-1H-tetrazole-5-thiol;
3-(6-Phenyl-thieno[3,2-d]pyrimidin-4-ylamino)-benzenesulfonamide;
(2-Methyl-benzothiazol-6-yl)-(6-phenyl-thieno[3,2-d]pyrimidin-4-yl)-amine;
[4-(Morpholine-4-sulfonyl)-phenyl]-(6-phenyl-thieno[3,2-d]pyrimidin-4-yl)-amine;
[3,5-Dimethyl-4-(thiophen-3-yl methoxy )-phenyl]-(6-phenyl-thieno[3,2-d]pyrimidin-4-yl)-amine;
[4,5-Dimethoxy-2-(1H-tetrazol-5-yl)-phenyl]-(6-phenyl-thieno[3,2-d]pyrimidin-4-yl)-amine;
5-[4-(6-Phenyl-thieno[3,2-d]pyrimidin-4-ylamino)-phenyl]-oxazolidine-2,4-dione;
1-Ethyl-5-(6-phenyl-thieno[3,2-d]pyrimidin-4-ylamino)-1,3-dihydro-indol-2-one;
6-(6-Phenyl-thieno[3,2-d]pyrimidin-4-ylamino)-3H-benzooxazol-2-one;
Dibenzothiophen-4-yl-(6-phenyl-thieno[3,2-d]pyrimidin-4-yl)-amine;
N-(6-Phenyl-thieno[3,2-d]pyrimidin-4-yl)-Nxe2x80x2-p-tolyl-benzene-1,2-diamine;
(2-Furan-2-yl-1-methyl-1H-benzoimidazol-5-yl)-(6-phenyl-thieno[3,2-d]pyrimidin-4-yl)-amine;
5-(6-Phenyl-thieno[3,2-d]pyrimidin-4-ylamino)-benzo[b]thiophene-2-carbonitrile;
(6-Phenyl-thieno[3,2-d]pyrimidin-4-yl)-(2-pyridin-4-yl-1H-benzoimidazol-5-yl)-amine;
[4-(1-Methyl-1H-imidazol-2-ylsulfanyl)-phenyl]-(6-phenyl-thieno[3,2-d]pyrimidin-4-yl)-amine;
(6-Phenyl-thieno[3,2-d]pyrimidin-4-yl)-[4-(pyridin-2-yloxy)-phenyl]-amine;
[4-(5-Methyl-tetrazol-1-yl)-phenyl]-(6-phenyl-thieno[3,2-d]pyrimidin-4-yl)-amine;
1-[3-(6-Phenyl-thieno[3,2-d]pyrimidin-4-ylamino)-phenyl]-1H-tetrazole-5-thiol;
4-[4-(6-Phenyl-thieno[3,2-d]pyrimidin-4-ylamino)-phenylamino]-phenol;
[3-(3-Methyl4,5-dihydro-pyrazol-1-yl)-phenyl]-(6-phenyl-thieno[3,2-d]pyrimidin-4-yl)-amine;
Benzo[1,2,3]thiadiazol-6-yl-(6-phenyl-thieno[3,2-d]pyrimidin-4-yl)-amine;
4-[7-(2-Methyl-1H-indol-5-ylamino)-thieno[3,2-b]pyridin-2-yl]-benzaldehyde
[6-(4-Fluoro-phenyl)-thieno[3,2-d]pyrimidin-4-yl]-(1H-indol-5-yl)-amine;
and the pharmaceutically acceptable salts and hydrates of the foregoing compounds. The present invention also relates to intermediate compounds of the formulas 25 and 26 
and to pharmaceutically acceptable salts thereof, wherein:
wherein X1 is N or CH;
Z1 is halo and Z2 is xe2x80x94NR1R2; or
Z1 is R11 and Z2 is halo; or
Z1 and Z2 are each independently halo;
R1 is H, C1-C6 alkyl or xe2x80x94C(O)(C1-C6 alkyl);
R2 is C6-C10 aryl or 5-13 membered heterocyclic, wherein said R2 groups are optionally substituted by 1 to 5 R5 substituents,
each R5 is independently selected from halo, cyano, nitro, trifluoromethoxy, trifluoromethyl, azido, xe2x80x94C(O)R8, xe2x80x94C(O)OR8, xe2x80x94OC(O)R8, xe2x80x94OC(O)OR8, xe2x80x94NR6C(O)R7, xe2x80x94C(O)NR6R7, xe2x80x94NR6R7, xe2x80x94OR9, xe2x80x94SO2NR6R7, C1-C6 alkyl, xe2x80x94(CH2)jO(CH2)qNR6R7, xe2x80x94(CH2)tO(CH2)qOR9, xe2x80x94(CH2)tOR9, xe2x80x94S(O)j(C1-C6 alkyl), xe2x80x94(CH2)t(C6-C10 aryl), xe2x80x94(CH2)t(5-10 membered heterocyclic), xe2x80x94C(O)(CH2)t(C6-C10 aryl), xe2x80x94(CH2)tO(CH2)j(C6-C10 aryl), xe2x80x94(CH2)tO(CH2)q(5-10 membered heterocyclic xe2x80x94C(O)(CH2)t(5-10 membered heterocyclic), xe2x80x94(CH2)jNR7(CH2)qNR6R7, xe2x80x94(CH2)jNR7CH2C(O)NR6R7, xe2x80x94(CH2)jNR7(CH2)qNR9C(O)R8, xe2x80x94(CH2)jNR7(CH2)tO(CH2)qOR9, xe2x80x94(CH2)jNR7(CH2)qS(O)j(C1-C6 alkyl), xe2x80x94(CH2)jNR7(CH2)tR6, xe2x80x94SO2(CH2)t(C6-C10 aryl), and xe2x80x94SO2(CH2)t(5-10 membered heterocyclic), wherein j is an integer ranging from 0 to 2, t is an integer ranging from 0 to 6, q is an integer ranging from 2 to 6, the xe2x80x94(CH2)qxe2x80x94, and xe2x80x94(CH2)txe2x80x94 moieties of the foregoing R5 groups optionally include a carbon-carbon double or triple bond where t is an integer between 2 and 6, and the alkyl, aryl and heterocyclic moieties of the foregoing R5 groups are optionally substituted by 1 to 3 substituents independently selected from halo, cyano, nitro, trifluoromethyl, azido, xe2x80x94C(O)R8, xe2x80x94C(O)OR8, xe2x80x94OC(O)R8, xe2x80x94OC(O)OR8, xe2x80x94NR6C(O)R7, xe2x80x94C(O)NR6R7, xe2x80x94(CH2)tNR6R7, C1-C6 alkyl, xe2x80x94(CH2)t(C6-C10 aryl), xe2x80x94(CH2)t(5-10 membered heterocyclic), xe2x80x94(CH2)tO(CH2)qOR9, and xe2x80x94(CH2)tOR9, wherein t is an integer ranging from 0 to 6 and q is an integer ranging from 2 to 6;
each R6 and R7 is independently selected from H, C1-C6 alkyl, xe2x80x94(CH2)t(C6-C10 aryl), xe2x80x94(CH2)t(5-10 membered heterocyclic), xe2x80x94(CH2)tO(CH2)qOR9, and xe2x80x94(CH2)tOR9, wherein t is an integer ranging from 0 to 6 and q is an integer ranging from 2 to 6, and the alkyl, aryl and heterocyclic moieties of the foregoing R6 and R7 groups are optionally substituted by 1 to 3 substituents independently selected from halo, cyano, nitro, trifluoromethyl, azido, xe2x80x94C(O)R8, xe2x80x94C(O)OR8, xe2x80x94CO(O)R8, xe2x80x94OC(O)OR8, xe2x80x94NR9C(O)R10, xe2x80x94C(O)NR9R10, xe2x80x94NR9R10, C1-C6 alkyl, xe2x80x94(CH2)t(C6-C10 aryl), xe2x80x94(CH2)t(5-10 membered heterocyclic), xe2x80x94(CH2)tO(CH2)qOR9, and xe2x80x94(CH2)tOR9, wherein t is an integer ranging from 0 to 6 and q is an integer ranging from 2 to 6, with the proviso that where R6 and R7 are both attached to the same nitrogen, then R6 and R7 are not both bonded to the nitrogen directly through an oxygen;
each R8 is independently selected from H, C1-C10 alkyl, xe2x80x94(CH2)t(C6-C10 aryl), and xe2x80x94(CH2)t(5-10 membered heterocyclic), wherein t is an integer ranging from 0 to 6;
each R9 and R10 is independently selected from H and C1-C6 alkyl; and,
R11 is H, C1-C6 alkyl, xe2x80x94C(O)NR6R9, xe2x80x94C(O)(C6-C10 aryl), xe2x80x94(CH2)t(C6-C10 aryl), or xe2x80x94(CH2)t(5-10 membered heterocyclic), wherein t is an integer ranging from 0 to 6, wherein said R11 groups, other than H, are optionally substituted by tert-butyl-dimethyl-silanyl and 1 to 3 R5 groups.
The above intermediates of formulas 25 and 26 may be used to prepare the above compounds of formulas 1 and 2.
The invention also relates to a pharmaceutical composition for the treatment of a hyperproliferative disorder in a mammal which comprises a therapeutically effective amount of a compound of formula 1 or 2, or a pharmaceutically acceptable salt or hydrate thereof, and a pharmaceutically acceptable carrier. In one embodiment, said pharmaceutical composition is for the treatment of cancer such as brain, lung, squamous cell, bladder, gastric, pancreatic, breast, head, neck, renal, kidney, ovarian, prostate, colorectal, oesophageal, gynecological or thyroid cancer. In another embodiment, said pharmaceutical composition is for the treatment of a non-cancerous hyperproliferative disorder such as benign hyperplasia of the skin (e.g., psoriasis) or prostate (e.g., benign prostatic hypertropy (BPH)).
The invention also relates to a pharmaceutical composition for the treatment of pancreatitis or kidney disease (including proliferative glomerulonephritis and diabetes-induced renal disease) in a mammal which comprises a therapeutically effective amount of a compound of formula 1 or 2, or a pharmaceutically acceptable salt or hydrate thereof, and a pharmaceutically acceptable carrier.
The invention also relates to a pharmaceutical composition for the prevention of blastocyte implantation in a mammal which comprises a therapeutically effective amount of a compound of formula 1 or 2, or a pharmaceutically acceptable salt or hydrate thereof, and a pharmaceutically acceptable carrier.
The invention also relates to a pharmaceutical composition for treating a disease related to vasculogenesis or angiogenesis in a mammal which comprises a therapeutically effective amount of a compound of formula 1 or 2, or a pharmaceutically acceptable salt or hydrate thereof, and a pharmaceutically acceptable carrier. In one embodiment, said pharmaceutical composition is for treating a disease selected from the group consisting of tumor angiogenesis, chronic inflammatory disease such as rheumatoid arthritis, atherosclerosis, skin diseases such as psoriasis, excema, and scleroderma, diabetes, diabetic retinopathy, retinopathy of prematurity, age-related macular degeneration, hemangioma, glioma, melanoma, Kaposi""s sarcoma and ovarian, breast, lung, pancreatic, prostate, colon and epidermoid cancer.
The invention also relates to a method of treating a hyperproliferative disorder in a mammal which comprises administering to said mammal a therapeutically effective amount of the compound of formula 1 or 2, or a pharmaceutically acceptable salt or hydrate thereof. In one embodiment, said method relates to the treatment of cancer such as brain, squamous cell, bladder, gastric, pancreatic, breast, head, neck, oesophageal, prostate, colorectal, lung, renal, kidney, ovarian, gynecological or thyroid cancer. In another embodiment, said method relates to the treatment of a non-cancerous hyperproliferative disorder such as benign hyperplasia of the skin (e.g., psoriasis) or prostate (e.g., benign prostatic hypertropy (BPH)).
The invention also relates to a method for the treatment of a hyperproliferative disorder in a mammal which comprises administering to said mammal a therapeutically effective amount of a compound of formula 1 or 2, or a pharmaceutically acceptable salt or hydrate thereof, in combination with an anti-tumor agent selected from the group consisting of mitotic inhibitors, alkylating agents, anti-metabolites, intercalating antibiotics, growth factor inhibitors, cell cycle inhibitors, enzymes, topoisomerase inhibitors, biological response modifiers, anti-hormones, and anti-androgens.
The invention also relates to a method of treating pancreatitis or kidney disease in a mammal which comprises administering to said mammal a therapeutically effective amount of a compound of formula 1 or 2, or a pharmaceutically acceptable salt or hydrate thereof.
The invention also relates to a method of preventing blastocyte implantation in a mammal which comprises administering to said mammal a therapeutically effective amount of a compound of formula 1 or 2, or a pharmaceutically acceptable salt or hydrate thereof.
The invention also relates to a method of treating diseases related to vasculogenesis or angiogenesis in a mammal which comprises administering to said mammal an effective amount of a compound of formula 1 or 2, or a pharmaceutically acceptable salt or hydrate thereof. In one embodiment, said method is for treating a disease selected from the group consisting of tumor angiogenesis, chronic inflammatory disease such as rheumatoid arthritis, atherosclerosis, skin diseases such as psoriasis, excema, and scleroderma, diabetes, diabetic retinopathy, retinopathy of prematurity, age-related macular degeneration, hemangioma, glioma, melanoma, Kaposi""s sarcoma and ovarian, breast, lung, pancreatic, prostate, colon and epidermoid cancer.
Patients that can be treated with the compounds of formulas 1 and 2, and the pharmaceutically acceptable salts and hydrates of said compounds, according to the methods of this invention include, for example, patients that have been diagnosed as having psoriasis, BPH, lung cancer, bone cancer, pancreatic cancer, skin cancer, cancer of the head and neck, cutaneous or intraocular melanoma, uterine cancer, ovarian cancer, rectal cancer, cancer of the anal region, stomach cancer, colon cancer, breast cancer, gynecologic tumors (e.g., uterine sarcomas, carcinoma of the fallopian tubes, carcinoma of the endometrium, carcinoma of the cervix, carcinoma of the vagina or carcinoma of the vulva), Hodgkin""s disease, cancer of the esophagus, cancer of the small intestine, cancer of the endocrine system (e.g., cancer of the thyroid, parathyroid or adrenal glands), sarcomas of soft tissues, cancer of the urethra, cancer of the penis, prostate cancer, chronic or acute leukemia, solid tumors of childhood, lymphocytic lymphonas, cancer of the bladder, cancer of the kidney or ureter (e.g., renal cell carcinoma, carcinoma of the renal pelvis), or neoplasms of the central nervous system (e.g., primary CNS lymphona, spinal axis tumors, brain stem gliomas or pituitary adenomas).
The term xe2x80x9chaloxe2x80x9d, as used herein, unless otherwise indicated, means fluoro, chloro, bromo or iodo. Preferred halo groups are fluoro, chloro and bromo.
The term xe2x80x9calkylxe2x80x9d, as used herein, unless otherwise indicated, includes saturated monovalent hydrocarbon radicals having straight, cyclic or branched moieties. Said xe2x80x9calkylxe2x80x9d group may include an optional carbon-carbon double or triple bond where said alkyl group comprises at least two carbon atoms. It is understood that for cyclic moieties at least three carbon atoms are required in said alkyl group.
The term xe2x80x9calkoxyxe2x80x9d, as used herein, unless otherwise indicated, includes O-alkyl groups wherein xe2x80x9calkylxe2x80x9d is as defined above.
The term xe2x80x9carylxe2x80x9d, as used herein, unless otherwise indicated, includes an organic radical derived from an aromatic hydrocarbon by removal of one hydrogen, such as phenyl or naphthyl.
The term xe2x80x9c5-10 membered heterocyclicxe2x80x9d or xe2x80x9c5-13 membered heterocyclicxe2x80x9d, as used herein, unless otherwise indicated, includes aromatic and non-aromatic heterocyclic groups containing one to four heteroatoms each selected from O, S and N, wherein each heterocyclic group has from 5-10 or 5-13 atoms in its ring system. The heterocyclic groups include benzo-fused ring systems and ring systems substituted with one or two oxo (xe2x95x90O) moieties such as pyrrolidin-2-one. An example of a 5 membered heterocyclic group is thiazolyl, an example of a 10 membered heterocyclic group is quinolinyl, and an example of a 13 membered heterocyclic group is a carbazole group. Examples of non-aromatic heterocyclic groups are pyrrolidinyl, piperidino, morpholino, thiomorpholino and piperazinyl. Examples of aromatic heterocyclic groups are pyridinyl, imidazolyl, pyrimidinyl, pyrazolyl, triazolyl, pyrazinyl, tetrazolyl, furyl, thienyl, isoxazolyl and thiazolyl. Heterocyclic groups having a fused benzene ring include benzimidazolyl, benzofuranyl, and benzo[1,3]dioxolyl.
The phrase xe2x80x9cpharmaceutically acceptable salt(s)xe2x80x9d, as used herein, unless otherwise indicated, includes salts of acidic or basic groups which may be present in the compounds of formulas 1 and 2. The compounds of formulas 1 and 2 that are basic in nature are capable of forming a wide variety of salts with various inorganic and organic acids. The acids that may be used to prepare pharmaceutically acceptable acid addition salts of such basic compounds of formulas 1 and 2 are those that form non-toxic acid addition salts, i.e., salts containing pharmacologically acceptable anions, such as the hydrochloride, hydrobromide, hydroiodide, nitrate, sulfate, bisulfate, phosphate, acid phosphate, isonicotinate, acetate, lactate, salicylate, citrate, acid citrate, tartrate, pantothenate, bitartrate, ascorbate, succinate, maleate, gentisinate, fumarate, gluconate, glucaronate, saccharate, formate, benzoate, glutamate, methanesulfonate, ethanesulfonate, benzenesulfonate, p-toluenesulfonate and pamoate [i.e., 1,1xe2x80x2-methylene-bis-(2-hydroxy-3-naphthoate)] salts.
Those compounds of the formulas 1 and 2 that are acidic in nature, are capable of forming base salts with various pharmacologically acceptable cations. Examples of such salts include the alkali metal or alkaline earth metal salts and particularly, the sodium and potassium salts.
Certain compounds of formulas 1 and 2 may have asymmetric centers and therefore exist in different enantiomeric forms. This invention relates to the use of all optical isomers and stereoisomers of the compounds of formulas 1 and 2 and mixtures thereof. The compounds of formulas 1 and 2 may also exist as tautomers. This invention relates to the use of all such tautomers and mixtures thereof. 
The preparation of the compounds of the present invention is illustrated in the following Schemes 1-3.
The compounds of the present invention are readily prepared according to synthetic methods familiar to those skilled in the art. Scheme 1 illustrates a general synthetic procedure for preparing the compounds of the present invention. While Scheme 1 specifically illustrates the preparation of compounds of formula 1, it applies equally to the preparation of compounds of formula 2. The compound of formula 7 (in which X1 is as defined above) may be prepared by one or more procedures described in published PCT international applications numbers WO 95/19774 (published Jul. 27, 1995), WO 95/19970 (published Jul. 27, 1995), and WO 97/13771 (published Apr. 17, 1997). In addition, 4-chlorothieno[3,2-d]pyrimidine is commercially available, such as from Maybridge Chemical Co. Ltd. A preferred method of preparing 4-chlorothieno[3,2-d]pyridine is described below with reference to steps 1-3 of Scheme 2. In step 1 of Scheme 1, the compound of formula 7 may be converted to the corresponding bromo derivative of formula 8 by treating the starting compound with lithium diisopropylamine or n-butyllithium, and then 1,2-dibromo-1,1,2,2-tetrafluoroethane or bromine in a non-polar solvent, such as tetrahydrofuran (THF), at a temperature of about xe2x88x9278xc2x0 C. for a period of about 15 minutes to one-half hour and then gradually warming the mixture to room temperature (20-25xc2x0 C.). In step 2 of Scheme 1, the compound of formula 8 may be coupled with a compound of formula HNR1R2, wherein R1 and R2 are as defined above, optionally in the presence of a base, such as pyridine, triethylamine or sodium hydride, and optionally in the presence of pyridine hydrochloride as a catalyst, under an inert atmosphere, such as dry nitrogen gas, in a solvent, such as a C1-C6 alcohol, dimethylformamide (DMF), 1,2-dichloroethane (DCE), N-methylpyrrolidin-2-one (NMP), chloroform, acetonitrile, THF, dimethylsulfoxide (DMSO), 1,4-dioxane or pyridine, or a mixture of two or more of the foregoing solvents, preferably a mixture of t-butyl alcohol and DCE, at a temperature of from ambient to reflux temperature, preferably 80-125xc2x0 C., for a period of about 2 hours to 72 hours to provide the compound of formula 9. The foregoing reaction is preferably done in a sealed tube.
Where the compound of formula HNR1R2 is an optionally substituted indole or indoline moiety, such compounds can be prepared according to one or more methods known to those skilled in the art. Such methods are described in PCT international patent application publication number WO 95/23141, referred to above, and in W. C. Sumpter and F. M. Miller, xe2x80x9cHeterocyclic Compounds with Indole and Carbazole Systems,xe2x80x9d in volume 8 of xe2x80x9cThe Chemistry of Heterocyclic Compoundsxe2x80x9d, Interscience Publishers Inc., New York (1954). Optional substituents can be included as appropriate before or after the coupling step illustrated in Scheme 1. Prior to the coupling step, primary and secondary amino moieties (other than said amine of formula HNR1R2) are preferably protected using a nitrogen protecting group known to those skilled in the art. Such protecting groups and their use are described in T. W. Greene and P. G. M. Wuts, xe2x80x9cProtective Groups in Organic Synthesis,xe2x80x9d Second Edition, John Wiley and Sons, New York, 1991.
In step 3 of Scheme 1, the compound of formula 9 may be converted to the compound of formula 1 by coupling the starting compound with a compound of the formula R11xe2x80x94B(OH)2 (wherein R11 is as defined above) in the presence of 1,4-bis(diphenylphosphino)butane and a palladium catalyst, such as bis(benzonitrile)-palladium(II) chloride, a base, such as sodium or potassium carbonate, and a solvent, such as toluene, ethanol, THF, DMF, or dimethoxyethane (DME), preferably a mixture of toluene, ethanol and THF, at a temperature within the range of about 50-110xc2x0 C. for a period of about 1 to 24 hours. This step is analogous to the Suzuki coupling procedure described in N. Miyaura, A. Suzuki, Chem. Rev. 1995, 95, 2457. In the alternative, steps 2 and 3 of Scheme 1 may be reversed. That is, the R11 group may be introduced into the compound of formula 7 followed by the coupling of the resulting compound with the compound of formula HNR1R2 as described above. In another procedure, step 3 of Scheme 1 may be achieved by reacting the compound of formula 9 with a compound of the formula (trialkylstannyl)-R11 (wherein R11 is as defined above), such as (tributylstannyl)-R11, in the presence of copper iodide and trans-benzyl(chloro)bis(triphenylphosphine)palladium(II) in DMF at a temperature of about 90xc2x0 C. for a period of about 14 hours. The starting compound for this procedure, specifically (tributylstannyl)-R11, may be prepared from R11xe2x80x94Br by at least three separate procedures. In a first procedure, R11xe2x80x94Br may be treated with (tributylstannyl)-chloride and n-butyllithium in THF or DMF to provide (tributylstannyl)-R11. In a second procedure, R11xe2x80x94Br may be treated with Bu3Snxe2x80x94SnBu3, wherein Bu represents butyl, and sodium metal to provide (tributylstannyl)-R11. And in a third procedure, R11xe2x80x94Br may be treated with Bu3Snxe2x80x94SnBu3, wherein Bu represents butyl, and Pd(PPh3)4, wherein Ph represents phenyl, in toluene to provide (tributylstannyl)-R11.
Following or before step 3 of Scheme 1, the R11 group may be modified to introduce one or more R5 groups (wherein R5 is as defined above). In a one preferred method, where R11 is a phenyl group that includes an aldehyde group, the aldehyde may be converted to a preferred aminomethyl group. In this process, the starting compound that includes an aldehyde on the R11 group is reacted with an amine of the formula HNR6R7 (wherein R6 and R7 are as defined above) in the presence of a reducing agent, such as sodium cyanoborohydride or sodium borohydride, in a solvent comprising acetic acid and ethanol or methanol at a temperature in the range of 0-100xc2x0 C., preferably room temperature. This process converts the aldehyde to a moiety of the formula R6R7NCH2xe2x80x94. Other methods of modifying the compounds of formula 1 will be obvious to those skilled in the art. The compounds of formula 2 are prepared in an analogous manner.
Scheme 2 illustrates a procedure for preparing the compounds of formula 1 wherein X1 is CH. In step 1 of Scheme 2, the compound of formula 10 (3-amino-thiophene-2-carboxylic acid methyl ester) is dissolved in sodium hydroxide and refluxed for about 2 hours. The solution is then cooled to 0xc2x0 C. and acidified to pH 5 with concentrated HCl at which time a precipitate will form. The precipitate is separated and treated with propanol and oxalic acid, and the solution is stirred at about 38xc2x0 C. for approximately 45 minutes to provide the compound of formula 11 (thiophen-3-ylamine). In step 2 of Scheme 2, the compound of formula 11 is dissolved in triethyl orthoformate and stirred at room temperature until dissolution is complete. 2,2-Dimethyl-[1,3]dioxane-4,6-dione is then added portionwise at room temperature, with a precipitate forming upon completion of the addition. The mixture is then heated at 85xc2x0 C. overnight. The resulting precipitate, which is an intermediate (2,2-dimethyl-5-(thiophen-3-ylaminomethylene)-[1,3]dioxane-4,6-dione), is then separated and washed. The intermediate is added to dowtherm A (heated to 260xc2x0 C.), and the resulting mixture is heated for 30 minutes and then cooled to room temperature to provide the compound of formula 12. In step 3 of Scheme 2, the compound of formula 12 is added to oxalyl chloride in a mixture of methylene chloride and DMF and heated to reflux for approximately two hours to provide the compound of formula 13. The compound of formula 13 may be converted to the compound of formula 14 as described above with respect to step 1 of Scheme 1. The compound of formula 14 may be converted to the compound of formula 15 as described above with respect to step 2 of Scheme 1. The compound of formula 15 may be converted to the compound of formula 16 as described above with respect to step 3 of Scheme 1.
Scheme 3 illustrates an alternative method of coupling the R11 group represented by the compound of formula 18, wherein each X moiety is CH or N and R5 is as defined above, with the remainder the compound of formula 1 or 2, which remainder is illustrated as the compound of formula 17. While the compound of formula 17 corresponds in structure to the compound of formula 1, the procedure described with respect to Scheme 3 may be followed to prepare corresponding compounds of formula 2. In step 1 of Scheme 3, the compound of formula 17 is coupled with the compound of formula 18 in DMF in the presence of copper iodide and trans-benzyl(chloro)bis-(triphenylphosphine)palladium(II) at a temperature of about 90xc2x0 C. for about 14 hours to provide the compound of formula 19. The compound of formula 19 may then be coupled with the compound of formula HNR1R2, wherein R1 and R2 are as defined above, as described above with respect to step 2 of Scheme 1 to provide the compound of formula 20.
The compounds of the present invention may have asymmetric carbon atoms. Such diasteromeric mixtures can be separated into their individual diastereomers on the basis of their physical chemical differences by methods known to those skilled in the art, for example, by chromatography or fractional crystallization. Enantiomers can be separated by converting the enantiomeric mixtures into a diasteromeric mixture by reaction with an appropriate optically active compound (e.g., alcohol), separating the diastereomers and converting (e.g., hydrolyzing) the individual diastereomers to the corresponding pure enantiomers. All such isomers, including diastereomer mixtures and pure enantiomers are considered as part of the invention.
The compounds of formulas 1 and 2 that are basic in nature are capable of forming a wide variety of different salts with various inorganic and organic acids. Although such salts must be pharmaceutically acceptable for administration to animals, it is often desirable in practice to initially isolate the compound of formula 1 or 2 from the reaction mixture as a pharmaceutically unacceptable salt and then simply convert the latter back to the free base compound by treatment with an alkaline reagent and subsequently convert the latter free base to a pharmaceutically acceptable acid addition salt. The acid addition salts of the base compounds of this invention are readily prepared by treating the base compound with a substantially equivalent amount of the chosen mineral or organic acid in an aqueous solvent medium or in a suitable organic solvent, such as methanol or ethanol. Upon careful evaporation of the solvent, the desired solid salt is readily obtained. The desired acid salt can also be precipitated from a solution of the free base in an organic solvent by adding to the solution an appropriate mineral or organic acid.
Those compounds of formulas 1 and 2 that are acidic in nature, are capable of forming base salts with various pharmacologically acceptable cations. Examples of such salts include the alkali metal or alkaline-earth metal salts and particularly, the sodium and potassium salts. These salts are all prepared by conventional techniques. The chemical bases which are used as reagents to prepare the pharmaceutically acceptable base salts of this invention are those which form non-toxic base salts with the acidic compounds of formulas 1 and 2. Such non-toxic base salts include those derived from such pharmacologically acceptable cations as sodium, potassium calcium and magnesium, etc. These salts can easily be prepared by treating the corresponding acidic compounds with an aqueous solution containing the desired pharmacologically acceptable cations, and then evaporating the resulting solution to dryness, preferably under reduced pressure. Alternatively, they may also be prepared by mixing lower alkanolic solutions of the acidic compounds and the desired alkali metal alkoxide together, and then evaporating the resulting solution to dryness in the same manner as before. In either case, stoichiometric quantities of reagents are preferably employed in order to ensure completeness of reaction and maximum yields of the desired final product.
The compounds of the present invention are potent inhibitors of the erbB family of oncogenic and protooncogenic protein tyrosine kinases such as epidermal growth factor receptor (EGFR), erbB2, HER3, or HER4 and thus are all adapted to therapeutic use as antiproliferative agents (e.g., anticancer) in mammals, particularly in humans. The compounds of the present invention are also inhibitors of angiogenesis and/or vasculogenesis. In particular, the compounds of the present invention are useful in the prevention and treatment of a variety of human hyperproliferative disorders such as malignant and benign tumors of the liver, kidney, bladder, breast, gastric, ovarian, colorectal, prostate, pancreatic, lung, vulval, thyroid, hepatic carcinomas, sarcomas, glioblastomas, head and neck, and other hyperplastic conditions such as benign hyperplasia of the skin (e.g., psoriasis) and benign hyperplasia of the prostate (e.g., BPH). It is, in addition, expected that a compound of the present invention may possess activity against a range of leukemias and lymphoid malignancies.
The compounds of the present invention may also be useful in the treatment of additional disorders in which aberrant expression ligand/receptor interactions or activation or signalling events related to various protein tyrosine kinases, are involved. Such disorders may include those of neuronal, glial, astrocytal, hypothalamic, and other glandular, macrophagal, epithelial, stromal, and blastocoelic nature in which aberrant function, expression, activation or signalling of the erbB tyrosine kinases are involved. In addition, the compounds of the present invention may have therapeutic utility in inflammatory, angiogenic and immunologic disorders involving both identified and as yet unidentified tyrosine kinases that are inhibited by the compounds of the present invention.
The in vitro activity of the compounds of formulas 1 and 2 in inhibiting the receptor tyrosine kinase (and thus subsequent proliferative response, e.g., cancer) may be determined by the following procedure.
The activity of the compounds of formulas 1 and 2, in vitro, can be determined by the amount of inhibition of the phosphorylation of an exogenous substrate (e.g., LyS3-Gastrin or polyGluTyr (4:1) random copolymer (I. Posner et al., J. Biol. Chem. 267 (29), 20638-47 (1992)) on tyrosine by epidermal growth factor receptor kinase by a test compound relative to a control. Affinity purified, soluble human EGF receptor (96 ng) is obtained according to the procedure in G. N. Gill, W. Weber, Methods in Enzymology 146, 82-88 (1987) from A431 cells (American Type Culture Collection, Rockville, Md.) and preincubated in a microfuge tube with EGF (2 xcexcg/ml) in phosphorylation buffer+vanadate (PBV: 50 mM HEPES, pH 7.4; 125 mM NaCl; 24 mM MgCl2; 100 xcexcM sodium orthovanadate), in a total volume of 10 xcexcl, for 20-30 minutes at room temperature. The test compound, dissolved in dimethylsulfoxide (DMSO), is diluted in PBV, and 10 xcexcl is mixed with the EGF receptor/EGF mix, and incubated for 10-30 minutes at 30xc2x0 C. The phosphorylation reaction is initiated by addition of 20 xcexcl 33P-ATP/substrate mix (120 xcexcM Lys3-Gastrin (sequence in single letter code for amino acids, KKKGPWLEEEEEAYGWLDF), 50 mM Hepes pH 7.4, 40 xcexcM ATP, 2 xcexcCi xcex3-[33P]-ATP) to the EGFr/EGF mix and incubated for 20 minutes at room temperature. The reaction is stopped by addition of 10 xcexcl stop solution (0.5 M EDTA, pH 8; 2 mM ATP) and 6 xcexcl 2N HCl. The tubes are centrifuged at 14,000 RPM, 4xc2x0 C., for 10 minutes. 35 xcexcl of supernatant from each tube is pipetted onto a 2.5 cm circle of Whatman P81 paper, bulk washed four times in 5% acetic acid, 1 liter per wash, and then air dried. This results in the binding of substrate to the paper with loss of free ATP on washing. The [33P] incorporated is measured by liquid scintillation counting. Incorporation in the absence of substrate (e.g., lys3-gastrin) is subtracted from all values as a background and percent inhibition is calculated relative to controls without test compound present. Such assays, carried out with a range of doses of test compounds, allow the determination of an approximate IC50 value for the in vitro inhibition of EGFR kinase activity.
The activity of the compounds of formulas 1 and 2, in vivo, can be determined by the amount of inhibition of tumor growth by a test compound relative to a control. The tumor growth inhibitory effects of various compounds are measured according to the methods of Corbett T. H., et al. xe2x80x9cTumor Induction Relationships in Development of Transplantable Cancers of the Colon in Mice for Chemotherapy Assays, with a Note on Carcinogen Structurexe2x80x9d, Cancer Res., 35, 2434-2439 (1975) and Corbett, T. H., et al., xe2x80x9cA Mouse Colon-tumor Model for Experimental Therapyxe2x80x9d, Cancer Chemother. Rep. (Part 2)xe2x80x9d, 5, 169-186 (1975), with slight modifications. Tumors are induced in the left flank by s.c. injection of 1xc3x97106 log phase cultured tumor cells (human MDA-MB-468 breast or human HN5 head and neck carcinoma cells) suspended in 0.10 ml RPMI 1640. After sufficient time has elapsed for the tumors to become palpable (2-3 mm in diameter) the test animals (athymic mice) are treated with active compound (formulated by dissolution in DMSO typically at a concentration of 50 to 100 mg/mL followed by 1:9 dilution into saline or, alternatively, 1:9 dilution into 0.1% Pluronic(trademark) P105 in 0.9% saline) by the intraperitoneal (ip) or oral (po) routes of administration twice daily (i.e., every 12 hours) for 5 consecutive days. In order to determine an anti-tumor effect, the tumor is measured in millimeters with Vernier calipers across two diameters and the tumor size (mg) is calculated using the formula: Tumor weight=(lengthxc3x97[width]2)/2, according to the methods of Geran, R. I., et al. xe2x80x9cProtocols for Screening Chemical Agents and Natural Products Against Animal Tumors and Other Biological Systemsxe2x80x9d, Third Edition, Cancer Chemother. Rep., 3, 1-104 (1972). Results are expressed as percent inhibition, according to the formula: Inhibition (%)=(TuWcontrolxe2x88x92TuWtest)/TuWcontrolxc3x97100%. The flank site of tumor implantation provides reproducible dose/response effects for a variety of chemotherapeutic agents, and the method of measurement (tumor diameter) is a reliable method for assessing tumor growth rates.
Other methods of assessing the activity of the compounds of the present invention are referred to in PCT international application publication number WO 95/21613 (published Aug. 17, 1995) which incorporated herein by reference.
The in vitro activity of the compounds of formulas 1 and 2 in inhibiting the KDR/VEGF receptor may be determined by the following procedure.
The ability of the compounds of the present invention to inhibit tyrosine kinase activity may be measured using a recombinant enzyme in an assay that measures the ability of compounds to inhibit the phosphorylation of the exogenous substrate, polyGluTyr (PGT, Sigma(trademark), 4:1). The kinase domain of the human KDR/VEGF receptor (amino acids 805-1350) is expressed in Sf9 insect cells as a glutathione S-transferase (GST)-fusion protein using the baculovirus expression system. The protein is purified from the lysates of these cells using glutathione agarose affinity columns. The enzyme assay is performed in 96-well plates that are coated with the PGT substrate (0.625 xcexcg PGT per well). Test compounds are diluted in dimethylsulfoxide (DMSO), and then added to the PGT plates so that the final concentration of DMSO in the assay is 1.6% (v/v). The recombinant enzyme is diluted in phosphorylation buffer (50 mM Hepes, pH 7.3, 125 mM NaCl, 24 mM MgCl2). The reaction is initiated by the addition of ATP to a final concentration of 10 xcexcM. After a 30 minute incubation at room temperature with shaking, the reaction is aspirated, and the plates are washed with wash buffer (PBS-containing 0.1% Tween-20). The amount of phosphorylated PGT is quantitated by incubation with a HRP-conjugated (HRP is horseradish peroxidase) PY-54 antibody (Transduction Labs), developed with TMB peroxidase (TMB is 3,3xe2x80x2,5,5xe2x80x2-tetramethylbenzene), and the reaction is quantitated on a BioRad(trademark) Microplate reader at 450 nM. Inhibition of the kinase enzymatic activity by the test compound is detected as a reduced absorbance, and the concentration of the compound that is required to inhibit the signal by 50% is reported as the IC50 value for the test compound.
To measure the ability of the compounds to inhibit KDR tyrosine kinase activity for the full length protein that exists in a cellular context, the porcine aortic endothelial (PAE) cells transfected with the human KDR (Waltenberger et al., J. Biol. Chem. 269:26988, 1994) may be used. Cells are plated and allowed to attach to 96-well dishes in the same media (Ham""s F12) with 10% FBS (fetal bovine serum). The cells are then washed, re-fed with serum depleted media that contains 0.1% (v/v) bovine serum albumin (BSA), and allowed to incubate for 24 hours. Immediately prior to dosing with compound, the cells are re-fed with the serum depleted media (without BSA). Test compounds, dissolved in DMSO, are diluted into the media (final DMSO concentration 0.5% (v/v)). At the end of a 2 hour incubation, VEGF165 (50 ng/ml final) is added to the media for an 8 minute incubation. The cells are washed and lysed in HNTG buffer (20 mM Hepes, pH 7.5, 150 mM NaCl, 0.2% Triton(trademark) X-100, 10% glycerol, 0.2 mM PMSF (phenymethylsulfonyl fluoride), 1 xcexcg/ml pepstatin, 1 xcexcg/ml leupeptin, 1 xcexcg/ml aprotonin, 2 mM sodium pyrophosphate, 2 mM sodium orthovanadate). The extent of phosphorylation of KDR is measured using an ELISA assay. The 96-well plates are coated with 1 xcexcg per well of goat anti-rabbit antibody. Unbound antibody is washed off the plate and remaining sites are blocked with Superblock buffer (Pierce) prior to addition of the anti-flk-1 C-20 antibody (0.5 xcexcg per plate, Santa Cruz). Any unbound antibody is washed off the plates prior to addition of the cell lysate. After a 2 hour incubation of the lysates with the flk-1 antibody, the KDR associated phosphotyrosine is quantitated by development with the HRP-conjugated PY-54 antibody and TMB, as described above. The ability of the compounds to inhibit the VEGF-stimulated autophosphorylation reaction by 50%, relative to VEGF-stimulated controls is reported as the IC50 value for the test compound.
The ability of the compounds to inhibit mitogenesis in human endothelial cells is measured by their ability to inhibit 3H-thymidine incorporation into HUVE cells (human umbilical vein endothelial cells, Clonetics(trademark)). This assay has been well described in the literature (Waltenberger J et al. J. Biol. Chem. 269: 26988, 1994; Cao Y et al. J. Biol. Chem. 271: 3154, 1996). Briefly, 104 cells are plated in collagen-coated 24-well plates and allowed to attach. Cells are re-fed in serum-free media, and 24 hours later are treated with various concentrations of compound (prepared in DMSO, final concentration of DMSO in the assay is 0.2% v/v), and 2-30 ng/ml VEGF165. During the last 3 hours of the 24 hour compound treatment, the cells are pulsed with 3H thymidine (NEN, 1 xcexcCi per well). The media are then removed, and the cells washed extensively with ice-cold Hank""s balanced salt solution, and then 2 times with ice cold trichloroacetic acid (10% v/v). The cells are lysed by the addition of 0.2 ml of 0.1 N NaOH, and the lysates transferred into scintillation vials. The wells are then washed with 0.2 ml of 0.1 N HCl, and this wash is then transferred to the vials. The extent of 3H thymidine incorporation is measured by scintillation counting. The ability of the compounds to inhibit incorporation by 50%, relative to control (VEGF treatment with DMSO vehicle only) is reported as the IC50 value for the test compound.
Administration of the compounds of the present invention (hereinafter the xe2x80x9cactive compound(s)xe2x80x9d) can be effected by any method that enables delivery of the compounds to the site of action. These methods include oral routes, intraduodenal routes, parenteral injection (including intravenous, subcutaneous, intramuscular, intravascular or infusion), topical, and rectal administration.
The amount of the active compound administered will be dependent on the subject being treated, the severity of the disorder or condition, the rate of administration and the judgement of the prescribing physician. However, an effective dosage is in the range of about 0.001 to about 100 mg per kg body weight per day, preferably about 1 to about 35 mg/kg/day, in single or divided doses. For a 70 kg human, this would amount to about 0.05 to about 7 g/day, preferably about 0.2 to about 2.5 g/day. In some instances, dosage levels below the lower limit of the aforesaid range may be more than adequate, while in other cases still larger doses may be employed without causing any harmful side effect, provided that such larger doses are first divided into several small doses for administration throughout the day.
The active compound may be applied as a sole therapy or may involve one or more other anti-tumour substances, for example those selected from, for example, mitotic inhibitors, for example vinblastine; alkylating agents, for example cis-platin, carboplatin and cyclophosphamide; anti-metabolites, for example 5-fluorouracil, cytosine arabinoside and hydroxyurea, or, for example, one of the preferred anti-metabolites disclosed in European Patent Application No. 239362 such as N-(5-[N-(3,4-dihydro-2-methyl-4-oxoquinazolin-6-ylmethyl)-N-methylamino]-2-thenoyl)L-glutamic acid; growth factor inhibitors; cell cycle inhibitors; intercalating antibiotics, for example adriamycin and bleomycin; enzymes, for example interferon; and anti-hormones, for example anti-estrogens such as Nolvadex(trademark) (tamoxifen) or, for example anti-androgens such as Casodex(trademark) (4xe2x80x2-cyano-3-(4-fluorophenylsulphonyl)-2-hydroxy-2-methyl-3xe2x80x2-(trifluoromethyl)propionanilide). Such conjoint treatment may be achieved by way of the simultaneous, sequential or separate dosing of the individual components of the treatment.
The pharmaceutical composition may, for example, be in a form suitable for oral administration as a tablet, capsule, pill, powder, sustained release formulations, solution, suspension, for parenteral injection as a sterile solution, suspension or emulsion, for topical administration as an ointment or cream or for rectal administration as a suppository. The pharmaceutical composition may be in unit dosage forms suitable for single administration of precise dosages. The pharmaceutical composition will include a conventional pharmaceutical carrier or excipient and a compound according to the invention as an active ingredient. In addition, it may include other medicinal or pharmaceutical agents, carriers, adjuvants, etc.
Exemplary parenteral administration forms include solutions or suspensions of active compounds in sterile aqueous solutions, for example, aqueous propylene glycol or dextrose solutions. Such dosage forms can be suitably buffered, if desired.
Suitable pharmaceutical carriers include inert diluents or fillers, water and various organic solvents. The pharmaceutical compositions may, if desired, contain additional ingredients such as flavorings, binders, excipients and the like. Thus for oral administration, tablets containing various excipients, such as citric acid may be employed together with various disintegrants such as starch, alginic acid and certain complex silicates and with binding agents such as sucrose, gelatin and acacia. Additionally, lubricating agents such as magnesium stearate, sodium lauryl sulfate and talc are often useful for tableting purposes. Solid compositions of a similar type may also be employed in soft and hard filled gelatin capsules. Preferred materials, therefor, include lactose or milk sugar and high molecular weight polyethylene glycols. When aqueous suspensions or elixirs are desired for oral administration the active compound therein may be combined with various sweetening or flavoring agents, coloring matters or dyes and, if desired, emulsifying agents or suspending agents, together with diluents such as water, ethanol, propylene glycol, glycerin, or combinations thereof.
Methods of preparing various pharmaceutical compositions with a specific amount of active compound are known, or will be apparent, to those skilled in this art. For examples, see Remington""s Pharmaceutical Sciences, Mack Publishing Company, Easter, Pa., 15th Edition (1975).